taq  (New England Biolabs)


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    Name:
    Taq I Methyltransferase
    Description:
    Taq I Methyltransferase 1 000 units
    Catalog Number:
    M0219S
    Price:
    76
    Category:
    DNA Methylases
    Size:
    1 000 units
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    Structured Review

    New England Biolabs taq
    Taq I Methyltransferase
    Taq I Methyltransferase 1 000 units
    https://www.bioz.com/result/taq/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    taq - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity"

    Article Title: Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1274

    ( A ) Schematic showing the use of PspOMI digestion to evaluate the retention rate of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside (see Table 4 for the sequence of oligonucleotides used). First, the Z -template (Temp-R-72- Z ) was amplified for 30 cycles in the presence of dNTPs, d Z TP and d P TP using Taq DNA polymerase. The final amplicon duplexes contained two kinds of products: one retaining the Z : P pair (product 2), the other misincorporating dC:dG pair (product 1). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 72-meroligonucleotides (full-length product, FLP) and all the oligonucleotides [including 72-merand 39-mer(digested fragment)] reflects the retention rate of Z : P pair in recognition sequence during PCR amplification. ( B ) Retention rates of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside with Taq DNA polymerase. The experiments were carried out according to the above schematic ( Figure 4 A). Lane 1 (control 1): PspOMI digestion of PCR product amplified by using the standard template (Temp-R-72-C). Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM). Lane 2 (control 2): misincorporation rate of PCR amplification of the standard template (Temp-R-72-C) in the presence of d Z TP and d P TP. Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM), d Z TP and d P TP (varying as indicated). Lanes 3–5: Retention rates of Z : P pair during the PCR amplification of Z -template (Temp-R-72- Z ). Final concentrations of PCR reaction mixture: dNTPs (200μM each), d Z TP and d P TP (varying as indicated). The concentration of the forward and reverse primers was fixed at 0.25 μM, while the concentration of templates were 250 pM (lane 3), 25 pM (lane 4) and 2.5 pM (lane 5), respectively.
    Figure Legend Snippet: ( A ) Schematic showing the use of PspOMI digestion to evaluate the retention rate of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside (see Table 4 for the sequence of oligonucleotides used). First, the Z -template (Temp-R-72- Z ) was amplified for 30 cycles in the presence of dNTPs, d Z TP and d P TP using Taq DNA polymerase. The final amplicon duplexes contained two kinds of products: one retaining the Z : P pair (product 2), the other misincorporating dC:dG pair (product 1). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 72-meroligonucleotides (full-length product, FLP) and all the oligonucleotides [including 72-merand 39-mer(digested fragment)] reflects the retention rate of Z : P pair in recognition sequence during PCR amplification. ( B ) Retention rates of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside with Taq DNA polymerase. The experiments were carried out according to the above schematic ( Figure 4 A). Lane 1 (control 1): PspOMI digestion of PCR product amplified by using the standard template (Temp-R-72-C). Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM). Lane 2 (control 2): misincorporation rate of PCR amplification of the standard template (Temp-R-72-C) in the presence of d Z TP and d P TP. Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM), d Z TP and d P TP (varying as indicated). Lanes 3–5: Retention rates of Z : P pair during the PCR amplification of Z -template (Temp-R-72- Z ). Final concentrations of PCR reaction mixture: dNTPs (200μM each), d Z TP and d P TP (varying as indicated). The concentration of the forward and reverse primers was fixed at 0.25 μM, while the concentration of templates were 250 pM (lane 3), 25 pM (lane 4) and 2.5 pM (lane 5), respectively.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Labeling, Concentration Assay

    ( A ) Schematic showing the use of PspOMI digestion to evaluate the misincorporation rate of Z and (or) P pair during the PCR amplifications of the standard template (see Table 3 for the sequence of oligonucleotides used). First, the standard template (Tem-R-81) were amplified for 30–40 PCR cycles in the presence of dNTPs (200μM each), d Z TP (25μM) and (or) d P TP (25μM). The final amplicon duplexes contained two kinds of products: one retaining the dC:dG pair (product 1), the other misincorporating Z : P pair (product 2). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 81-mer oligonucleotides (full-length product, FLP) and all the oligonucleotides [including 81-mer and 42-mer oligonucleotides (digested fragment)] represents the misincorporation rate of Z and (or) P in recognition sequence during PCR amplification. ( B ) Misincorporation rates of PCR amplification of the standard template in the presence of the AEGIS components using Deep Vent (exo + and exo − ) DNA polymerases at indicated pH values. Four parallel PCRs were performed to amply the standard template ( Table 3 ) containing a recognition sequence (5′-GGGCCC-3′), followed by digestion with PspOMI for 16 h. The ratio between the amount of full-length product (FLP) and all the oligonucleotides indicate the misincorporation rate and shown on the figure. Lane 1: negative control PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each), followed by digestion with PspOMI. Lane 2: five-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each) and d Z TP (25 μM), followed by digestion with PspOMI. Lane 3: five-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each) and d P TP (25 μM), followed by digestion with PspOMI. Lane 4: six-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each), d Z TP (25 μM) and d P TP (25 μM), followed by digestion with PspOMI. ( C ) Misincorporation rates of PCR amplification of the standard template in the presence of d Z TP and (or) d P TP using Taq and Phusion DNA polymerases at indicated pH values. The reactions followed the same protocol as in Figure 3 B except for the polymerases.
    Figure Legend Snippet: ( A ) Schematic showing the use of PspOMI digestion to evaluate the misincorporation rate of Z and (or) P pair during the PCR amplifications of the standard template (see Table 3 for the sequence of oligonucleotides used). First, the standard template (Tem-R-81) were amplified for 30–40 PCR cycles in the presence of dNTPs (200μM each), d Z TP (25μM) and (or) d P TP (25μM). The final amplicon duplexes contained two kinds of products: one retaining the dC:dG pair (product 1), the other misincorporating Z : P pair (product 2). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 81-mer oligonucleotides (full-length product, FLP) and all the oligonucleotides [including 81-mer and 42-mer oligonucleotides (digested fragment)] represents the misincorporation rate of Z and (or) P in recognition sequence during PCR amplification. ( B ) Misincorporation rates of PCR amplification of the standard template in the presence of the AEGIS components using Deep Vent (exo + and exo − ) DNA polymerases at indicated pH values. Four parallel PCRs were performed to amply the standard template ( Table 3 ) containing a recognition sequence (5′-GGGCCC-3′), followed by digestion with PspOMI for 16 h. The ratio between the amount of full-length product (FLP) and all the oligonucleotides indicate the misincorporation rate and shown on the figure. Lane 1: negative control PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each), followed by digestion with PspOMI. Lane 2: five-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each) and d Z TP (25 μM), followed by digestion with PspOMI. Lane 3: five-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each) and d P TP (25 μM), followed by digestion with PspOMI. Lane 4: six-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each), d Z TP (25 μM) and d P TP (25 μM), followed by digestion with PspOMI. ( C ) Misincorporation rates of PCR amplification of the standard template in the presence of d Z TP and (or) d P TP using Taq and Phusion DNA polymerases at indicated pH values. The reactions followed the same protocol as in Figure 3 B except for the polymerases.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Transmission Electron Microscopy, Amplification, Labeling, Negative Control

    2) Product Images from "A PCR-Based Method for Distinguishing between Two Common Beehive Bacteria, Paenibacillus larvae and Brevibacillus laterosporus"

    Article Title: A PCR-Based Method for Distinguishing between Two Common Beehive Bacteria, Paenibacillus larvae and Brevibacillus laterosporus

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01886-18

    Brevibacillus -specific primers for the rpoB gene discriminate between Paenibacillus and Brevibacillus bacteria and verify that field isolates (BL) are Brevibacillus laterosporus . Brevibacillus -specific primers (BL rpoB) were used to attempt PCR amplification of the rpoB gene found in B. laterosporus from various control strains as well as field isolates (BL). TAQ (New England BioLabs) PCR was performed according to the manufacturer's instructions with an annealing temperature of 69.1°C. Lanes were loaded as follows (the bacterial template is indicated): 1, B. laterosporus 40A1 (ATCC 9141); 2, B. brevis (ATCC 8246); 3, P. larvae (ATCC 9545); 4, P. alvei (BGSC 33A1); 5, P. polymyxa (BGSC 25A2); 6, Paenibacillus sp. (BGSC 35A1); 7, Br1; 8, Br3; 9, Br4; 10, Br5; 11, Br6; 12, Br12; 13, Brevibacillus sp.; 14, PL357. The black line indicates a separate gel. The ladder included in the far-left lane of each gel is a 1-kb Plus DNA ladder (Gold Biotechnology), with the top bright band representing 3 kb and the bottom bright band representing 1 kb.
    Figure Legend Snippet: Brevibacillus -specific primers for the rpoB gene discriminate between Paenibacillus and Brevibacillus bacteria and verify that field isolates (BL) are Brevibacillus laterosporus . Brevibacillus -specific primers (BL rpoB) were used to attempt PCR amplification of the rpoB gene found in B. laterosporus from various control strains as well as field isolates (BL). TAQ (New England BioLabs) PCR was performed according to the manufacturer's instructions with an annealing temperature of 69.1°C. Lanes were loaded as follows (the bacterial template is indicated): 1, B. laterosporus 40A1 (ATCC 9141); 2, B. brevis (ATCC 8246); 3, P. larvae (ATCC 9545); 4, P. alvei (BGSC 33A1); 5, P. polymyxa (BGSC 25A2); 6, Paenibacillus sp. (BGSC 35A1); 7, Br1; 8, Br3; 9, Br4; 10, Br5; 11, Br6; 12, Br12; 13, Brevibacillus sp.; 14, PL357. The black line indicates a separate gel. The ladder included in the far-left lane of each gel is a 1-kb Plus DNA ladder (Gold Biotechnology), with the top bright band representing 3 kb and the bottom bright band representing 1 kb.

    Techniques Used: Polymerase Chain Reaction, Amplification

    PL Vagabond primers discriminate between P. larvae and other closely related bacteria. (A and B) Paenibacillus -specific primers were used to attempt PCR amplification of a region of the genome found in various control strains as well as field isolates. TAQ (New England BioLabs) PCR was performed according to the manufacturer's instructions with an annealing temperature of 64°C (A) or 60°C (B). Lanes were loaded identically for the two gels as follows (the bacterial template is indicated): 1, B. laterosporus 40A1 (ATCC 9141); 2, Br1; 3, Br2; 4, Br3; 5, Br4; 6, Br5; 7, Br6; 8, Br7; 9, Br9; 10, Br14; 11, B. brevis (ATCC 8246); 12, P. larvae (ATCC 9545); 13, PL309; 14, PL325; 15, PL335; 16, PL337; 17, PL338; 18, PL344; 19, PL345; 20, PL357; 21, Paenibacillus sp. (BGSC 35A1); 22, P. alvei (BGSC 33A1); 23, P. polymyxa (BGSC 25A2); 24, negative control. The black line indicates a separate gel. The ladder included at the far left lane of each gel is a 1-kb DNA ladder (Gold Biotechnology), with the top bright band representing 3 kb and the bottom bright band representing 1 kb.
    Figure Legend Snippet: PL Vagabond primers discriminate between P. larvae and other closely related bacteria. (A and B) Paenibacillus -specific primers were used to attempt PCR amplification of a region of the genome found in various control strains as well as field isolates. TAQ (New England BioLabs) PCR was performed according to the manufacturer's instructions with an annealing temperature of 64°C (A) or 60°C (B). Lanes were loaded identically for the two gels as follows (the bacterial template is indicated): 1, B. laterosporus 40A1 (ATCC 9141); 2, Br1; 3, Br2; 4, Br3; 5, Br4; 6, Br5; 7, Br6; 8, Br7; 9, Br9; 10, Br14; 11, B. brevis (ATCC 8246); 12, P. larvae (ATCC 9545); 13, PL309; 14, PL325; 15, PL335; 16, PL337; 17, PL338; 18, PL344; 19, PL345; 20, PL357; 21, Paenibacillus sp. (BGSC 35A1); 22, P. alvei (BGSC 33A1); 23, P. polymyxa (BGSC 25A2); 24, negative control. The black line indicates a separate gel. The ladder included at the far left lane of each gel is a 1-kb DNA ladder (Gold Biotechnology), with the top bright band representing 3 kb and the bottom bright band representing 1 kb.

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control

    3) Product Images from "PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications"

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    Journal: Scientific Reports

    doi: 10.1038/srep05052

    Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .
    Figure Legend Snippet: Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

    Techniques Used: Binding Assay, Amplification, Polymerase Chain Reaction

    Primers that anneal far away from the repetitive DNA perform much better in amplifying the desired product. Taq DNA polymerase (NE Biolabs) was used in the PCR amplification of the indicated region of the pdTALE12 plasmid. PCR conditions are given in the supplementary material .
    Figure Legend Snippet: Primers that anneal far away from the repetitive DNA perform much better in amplifying the desired product. Taq DNA polymerase (NE Biolabs) was used in the PCR amplification of the indicated region of the pdTALE12 plasmid. PCR conditions are given in the supplementary material .

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation

    PCR fragments generated upon a typical PCR amplification from the pTAL2 vector with 12.5 TALE DNA-binding repeats. Plasmid map is shown in Supplementary Fig. 3 . Proofreading Pfu polymerase (Bioline) and normal Taq polymerase (NE Biolabs) were used in PCR amplification. PCR conditions are described in the supplementary material .
    Figure Legend Snippet: PCR fragments generated upon a typical PCR amplification from the pTAL2 vector with 12.5 TALE DNA-binding repeats. Plasmid map is shown in Supplementary Fig. 3 . Proofreading Pfu polymerase (Bioline) and normal Taq polymerase (NE Biolabs) were used in PCR amplification. PCR conditions are described in the supplementary material .

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Plasmid Preparation, Binding Assay

    Testing the generality of the model to other template DNAs with repetitive sequences. (A). GFP-coding sequences were cloned into the pBasicS1 vector and their integrity were checked by sequencing and restriction enzyme digestions. (B). PCR results obtained with primers 390 and 570. Taq DNA polymerase (NE Biolabs) was used in PCR amplification. The arrow indicates the sequenced artifact product which contained only one copy of the GFP lacking the filler sequence. See the supplementary material for PCR conditions.
    Figure Legend Snippet: Testing the generality of the model to other template DNAs with repetitive sequences. (A). GFP-coding sequences were cloned into the pBasicS1 vector and their integrity were checked by sequencing and restriction enzyme digestions. (B). PCR results obtained with primers 390 and 570. Taq DNA polymerase (NE Biolabs) was used in PCR amplification. The arrow indicates the sequenced artifact product which contained only one copy of the GFP lacking the filler sequence. See the supplementary material for PCR conditions.

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Amplification

    4) Product Images from "Enzymatic incorporation of a third nucleobase pair"

    Article Title: Enzymatic incorporation of a third nucleobase pair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm395

    Retention of the d Z :d P pair in products generated by 16.6 theoretical PCR cycles (using Taq ) as a function of pH. Left : Seven cycles of ‘analytical primer extension’ by Vent (exo − ) with dZTPαS, dPTP and dNTPs converts 5′- 32 P labeled primer (P-RS-S16) to FLP with a phosphorothioate linkage joining nucleotides 25 and 26 (arising via incorporation of dZTPαS opposite retained d P in the amplicon) at pH 8.5. Right : Digestion of FLP using Exo III (20 U, final concentration 0.5 U/µl), giving pause bands where an phosphorothioate linker is present, either from dZTPαS incorporation (expected between nucleotides 25 and 26) or from the synthetic primer (between nucleotides 16 and 17). Control (C) used synthetic templates (containing 100% d Z :d P pair at position 26). Lanes Rf51, Rf21 and Rf25 hold reference 51-, 21- and 25-mers. Noticeably stronger are Exo III digestion bands indicating greater retention of d Z :d P pair at pH 7.8 and 8.0 than at 7.5 or 8.5.
    Figure Legend Snippet: Retention of the d Z :d P pair in products generated by 16.6 theoretical PCR cycles (using Taq ) as a function of pH. Left : Seven cycles of ‘analytical primer extension’ by Vent (exo − ) with dZTPαS, dPTP and dNTPs converts 5′- 32 P labeled primer (P-RS-S16) to FLP with a phosphorothioate linkage joining nucleotides 25 and 26 (arising via incorporation of dZTPαS opposite retained d P in the amplicon) at pH 8.5. Right : Digestion of FLP using Exo III (20 U, final concentration 0.5 U/µl), giving pause bands where an phosphorothioate linker is present, either from dZTPαS incorporation (expected between nucleotides 25 and 26) or from the synthetic primer (between nucleotides 16 and 17). Control (C) used synthetic templates (containing 100% d Z :d P pair at position 26). Lanes Rf51, Rf21 and Rf25 hold reference 51-, 21- and 25-mers. Noticeably stronger are Exo III digestion bands indicating greater retention of d Z :d P pair at pH 7.8 and 8.0 than at 7.5 or 8.5.

    Techniques Used: Generated, Polymerase Chain Reaction, Labeling, Amplification, Concentration Assay

    Incorporation of dPTPαS (presented as its pure S p diastereoisomer) or dZTPαS (presented as a 1:1 diastereomeric mixture) opposite template d Z or d P (respectively) to generate oligonucleotide products that have phosphorothioate linkages resistant to exonuclease digestion. Left: Running start extensions (by Taq and 9°N DNA polymerases, as indicated) of 5′- 32 P labeled 21-mer primers having a phosphorothioate linkage between nucleotides 16 and 17 and Z-Temp and P-Temp. Right: Exo III (100 U, final concentration 2.5 U/µl) digestion of full-length products (FLP) containing phosphorothioate linkages (digestion times indicated). A 26-mer band indicates pausing of Exo III digestion at phosphorothioate linkage arising via incorporation of dPTPαS or dZTPαS opposite d Z or d P (both at positions 26 in their respective templates). This shows that both dPTPαS and dZTPαS were incorporated opposite their partners. A 17-mer band appears only if Exo III digests through the 25–26 linkage, either because misincorporation at an earlier step prevents the respective thiotriphosphates from incorporating a phosphorothioate linkage to join nucleotides 25 and 26, or because Exo III has digested through a 25-26 phosphorothioate linkage having an Exo III-sensitive S P stereochemistry. Digestion will pause at the phophorothioate joining nucleotides 16 and 17 arising from the chemically synthesized primer (and therefore having both R P and S P stereochemistries). Extra pausing bands (between 51- and 26-mers) suggest that 9°N incorporates small amounts of dZTPαS opposite template d G . Low-weight molecular products arise by digestion through the S P phosphorothioates joining nucleotides 16 and 17. Lanes P- Taq and P-9°N indicate that extension was done with Taq or 9°N (respectively), and dPTPαS (in both). Lanes Z- Taq and Z-9°N indicate that extension was done with Taq or 9°N (respectively), and dZTPαS (in both).
    Figure Legend Snippet: Incorporation of dPTPαS (presented as its pure S p diastereoisomer) or dZTPαS (presented as a 1:1 diastereomeric mixture) opposite template d Z or d P (respectively) to generate oligonucleotide products that have phosphorothioate linkages resistant to exonuclease digestion. Left: Running start extensions (by Taq and 9°N DNA polymerases, as indicated) of 5′- 32 P labeled 21-mer primers having a phosphorothioate linkage between nucleotides 16 and 17 and Z-Temp and P-Temp. Right: Exo III (100 U, final concentration 2.5 U/µl) digestion of full-length products (FLP) containing phosphorothioate linkages (digestion times indicated). A 26-mer band indicates pausing of Exo III digestion at phosphorothioate linkage arising via incorporation of dPTPαS or dZTPαS opposite d Z or d P (both at positions 26 in their respective templates). This shows that both dPTPαS and dZTPαS were incorporated opposite their partners. A 17-mer band appears only if Exo III digests through the 25–26 linkage, either because misincorporation at an earlier step prevents the respective thiotriphosphates from incorporating a phosphorothioate linkage to join nucleotides 25 and 26, or because Exo III has digested through a 25-26 phosphorothioate linkage having an Exo III-sensitive S P stereochemistry. Digestion will pause at the phophorothioate joining nucleotides 16 and 17 arising from the chemically synthesized primer (and therefore having both R P and S P stereochemistries). Extra pausing bands (between 51- and 26-mers) suggest that 9°N incorporates small amounts of dZTPαS opposite template d G . Low-weight molecular products arise by digestion through the S P phosphorothioates joining nucleotides 16 and 17. Lanes P- Taq and P-9°N indicate that extension was done with Taq or 9°N (respectively), and dPTPαS (in both). Lanes Z- Taq and Z-9°N indicate that extension was done with Taq or 9°N (respectively), and dZTPαS (in both).

    Techniques Used: Labeling, Concentration Assay, Synthesized

    Retention of the d Z :d P pair in products generated by the indicated number of theoretical rounds of PCR using Taq , Vent (exo − ) and Deep Vent (exo − ) at pH 8.0. Left : FLP are formed by extension by 9°N polymerase of 5′- 32 P labeled primer (P-RS-S16) with unlabeled primer (Z-RS) and dZTPαS, dPTP and dNTPs using PCR amplicons generated by Taq , Vent (exo − ) and DV (exo − ) as templates (shown in Figure 7 ). Right : PAGE (20%) resolution of products from Exo III (20 U, final concentration 0.5 U/µl) digestion of the FLP from Left . A band indicates presence of a phosphorothioate linkage, either arising through the incorporation of dZTPαS opposite d P at position 26, or from the synthetic primer joining nucleotidies 16 and 17, or through misincorporation of d Z :d P pair during PCR. Controls (lanes marked C) are: (i) C, incubation without 9°N; the absence of extended primer indicates successful removal of Taq polymerase (lane 1, Figure 7 ) by the QIAquick Nucleotide Remove Kit. (ii) C −, extension by 9°N using synthetic templates containing d Z and d P (at position 26) with standard (oxygen-containing) dZTP and dPTP; absent a phosphorothioate linkage in the extension, Exo III degradation gives a 17-mer due to presence of a phosphorothioate only from the synthetic primer. (iii) C +, extension using synthetic templates as before, but with dZTPαS, generating phosphorothioate linkage in the product, Exo III degradation gives a 26-mer due to the phosphorothioate linkage arising from the incorporation of dZTPαS opposite d P in the synthetic template. P ( 21 ) indicates the 21-mer, 5′- 32 P labeled primer (P-RS-S16).
    Figure Legend Snippet: Retention of the d Z :d P pair in products generated by the indicated number of theoretical rounds of PCR using Taq , Vent (exo − ) and Deep Vent (exo − ) at pH 8.0. Left : FLP are formed by extension by 9°N polymerase of 5′- 32 P labeled primer (P-RS-S16) with unlabeled primer (Z-RS) and dZTPαS, dPTP and dNTPs using PCR amplicons generated by Taq , Vent (exo − ) and DV (exo − ) as templates (shown in Figure 7 ). Right : PAGE (20%) resolution of products from Exo III (20 U, final concentration 0.5 U/µl) digestion of the FLP from Left . A band indicates presence of a phosphorothioate linkage, either arising through the incorporation of dZTPαS opposite d P at position 26, or from the synthetic primer joining nucleotidies 16 and 17, or through misincorporation of d Z :d P pair during PCR. Controls (lanes marked C) are: (i) C, incubation without 9°N; the absence of extended primer indicates successful removal of Taq polymerase (lane 1, Figure 7 ) by the QIAquick Nucleotide Remove Kit. (ii) C −, extension by 9°N using synthetic templates containing d Z and d P (at position 26) with standard (oxygen-containing) dZTP and dPTP; absent a phosphorothioate linkage in the extension, Exo III degradation gives a 17-mer due to presence of a phosphorothioate only from the synthetic primer. (iii) C +, extension using synthetic templates as before, but with dZTPαS, generating phosphorothioate linkage in the product, Exo III degradation gives a 26-mer due to the phosphorothioate linkage arising from the incorporation of dZTPαS opposite d P in the synthetic template. P ( 21 ) indicates the 21-mer, 5′- 32 P labeled primer (P-RS-S16).

    Techniques Used: Generated, Polymerase Chain Reaction, Labeling, Polyacrylamide Gel Electrophoresis, Concentration Assay, Incubation

    5) Product Images from "A Recurrent Stop-Codon Mutation in Succinate Dehydrogenase Subunit B Gene in Normal Peripheral Blood and Childhood T-Cell Acute Leukemia"

    Article Title: A Recurrent Stop-Codon Mutation in Succinate Dehydrogenase Subunit B Gene in Normal Peripheral Blood and Childhood T-Cell Acute Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000436

    SDHB mutations in genomic DNAs (gDNAs). A. Taq I RE digestion of PCR-amplified exon 2 shows no evidence of undigested mutant DNAs at 260 bp size (pointed by an arrow head). Taq I RE digestion products of the wild-type DNA co-migrate at sizes of 128 and 132 bps (denoted by a star). B. Gel electrophoresis of Taq I RE-digested (mutation) enrichment PCR ( e PCR) products shows variable amounts of mutant gDNAs at 233 bp size (pointed by an arrow head), which are confirmed to be primarily composed of c.136C > T by sequencing ( Table 1 ). Taq I digestion of wild-type e PCR products gives two bands at sizes, 101 and 132 bp (shown by a star). C. e PCR analyses of leukemic cell lines demonstrate the presence of mutant gDNA in the MOLT-4 cell line (pointed by an arrow head) but not in other leukemic cell lines in this experiment. D. Direct sequence analysis of a gDNA e PCR product shows selective enrichment of the R46X mutation. E, F. Sequence chromatograms of the heterozygous DNA mutations detected in two T-ALL cell lines are shown.
    Figure Legend Snippet: SDHB mutations in genomic DNAs (gDNAs). A. Taq I RE digestion of PCR-amplified exon 2 shows no evidence of undigested mutant DNAs at 260 bp size (pointed by an arrow head). Taq I RE digestion products of the wild-type DNA co-migrate at sizes of 128 and 132 bps (denoted by a star). B. Gel electrophoresis of Taq I RE-digested (mutation) enrichment PCR ( e PCR) products shows variable amounts of mutant gDNAs at 233 bp size (pointed by an arrow head), which are confirmed to be primarily composed of c.136C > T by sequencing ( Table 1 ). Taq I digestion of wild-type e PCR products gives two bands at sizes, 101 and 132 bp (shown by a star). C. e PCR analyses of leukemic cell lines demonstrate the presence of mutant gDNA in the MOLT-4 cell line (pointed by an arrow head) but not in other leukemic cell lines in this experiment. D. Direct sequence analysis of a gDNA e PCR product shows selective enrichment of the R46X mutation. E, F. Sequence chromatograms of the heterozygous DNA mutations detected in two T-ALL cell lines are shown.

    Techniques Used: Polymerase Chain Reaction, Amplification, Mutagenesis, Nucleic Acid Electrophoresis, Sequencing

    6) Product Images from "Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification"

    Article Title: Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096640

    ASA assay of Factor II prothrombin, Human gDNA (WHO International Standards) with R660V mutant. Allele-specific primers A and G result in a end match or mismatch depending on the chosen template. In red primer A is shown, in blue primer G. RFU = relative fluorescence units. A) ASA real time PCR curves of Factor II homogygote (genotype A/A) using Taq DNA polymerase, B) Platinum Taq DNA polymerase, or C) mutant R660V. D) ASA real time PCR curves of Factor II wild-type (genotype G/G), E) ASA real time PCR curves of Factor II heterozygote (genotype G/A), F) Agarose gel electrophoresis analysis of ASA PCR. M = Marker, A = Primer A, G = Primer G, wild-type (WT) indicates the used wild-type template, homo, the homozygote and hetero, the heterozygote template.
    Figure Legend Snippet: ASA assay of Factor II prothrombin, Human gDNA (WHO International Standards) with R660V mutant. Allele-specific primers A and G result in a end match or mismatch depending on the chosen template. In red primer A is shown, in blue primer G. RFU = relative fluorescence units. A) ASA real time PCR curves of Factor II homogygote (genotype A/A) using Taq DNA polymerase, B) Platinum Taq DNA polymerase, or C) mutant R660V. D) ASA real time PCR curves of Factor II wild-type (genotype G/G), E) ASA real time PCR curves of Factor II heterozygote (genotype G/A), F) Agarose gel electrophoresis analysis of ASA PCR. M = Marker, A = Primer A, G = Primer G, wild-type (WT) indicates the used wild-type template, homo, the homozygote and hetero, the heterozygote template.

    Techniques Used: Mutagenesis, Fluorescence, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    7) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.05.27.120238

    Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
    Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

    Techniques Used: Quantitative RT-PCR, Amplification

    TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.
    Figure Legend Snippet: TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.

    Techniques Used: Quantitative RT-PCR, Amplification

    8) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.0c00778

    Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
    Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

    Techniques Used: Quantitative RT-PCR, Incubation, Amplification

    Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
    Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

    Techniques Used: Quantitative RT-PCR, Amplification

    9) Product Images from "A Recurrent Stop-Codon Mutation in Succinate Dehydrogenase Subunit B Gene in Normal Peripheral Blood and Childhood T-Cell Acute Leukemia"

    Article Title: A Recurrent Stop-Codon Mutation in Succinate Dehydrogenase Subunit B Gene in Normal Peripheral Blood and Childhood T-Cell Acute Leukemia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000436

    SDHB mutations in genomic DNAs (gDNAs). A. Taq I RE digestion of PCR-amplified exon 2 shows no evidence of undigested mutant DNAs at 260 bp size (pointed by an arrow head). Taq I RE digestion products of the wild-type DNA co-migrate at sizes of 128 and 132 bps (denoted by a star). B. Gel electrophoresis of Taq I RE-digested (mutation) enrichment PCR ( e PCR) products shows variable amounts of mutant gDNAs at 233 bp size (pointed by an arrow head), which are confirmed to be primarily composed of c.136C > T by sequencing ( Table 1 ). Taq I digestion of wild-type e PCR products gives two bands at sizes, 101 and 132 bp (shown by a star). C. e PCR analyses of leukemic cell lines demonstrate the presence of mutant gDNA in the MOLT-4 cell line (pointed by an arrow head) but not in other leukemic cell lines in this experiment. D. Direct sequence analysis of a gDNA e PCR product shows selective enrichment of the R46X mutation. E, F. Sequence chromatograms of the heterozygous DNA mutations detected in two T-ALL cell lines are shown.
    Figure Legend Snippet: SDHB mutations in genomic DNAs (gDNAs). A. Taq I RE digestion of PCR-amplified exon 2 shows no evidence of undigested mutant DNAs at 260 bp size (pointed by an arrow head). Taq I RE digestion products of the wild-type DNA co-migrate at sizes of 128 and 132 bps (denoted by a star). B. Gel electrophoresis of Taq I RE-digested (mutation) enrichment PCR ( e PCR) products shows variable amounts of mutant gDNAs at 233 bp size (pointed by an arrow head), which are confirmed to be primarily composed of c.136C > T by sequencing ( Table 1 ). Taq I digestion of wild-type e PCR products gives two bands at sizes, 101 and 132 bp (shown by a star). C. e PCR analyses of leukemic cell lines demonstrate the presence of mutant gDNA in the MOLT-4 cell line (pointed by an arrow head) but not in other leukemic cell lines in this experiment. D. Direct sequence analysis of a gDNA e PCR product shows selective enrichment of the R46X mutation. E, F. Sequence chromatograms of the heterozygous DNA mutations detected in two T-ALL cell lines are shown.

    Techniques Used: Polymerase Chain Reaction, Amplification, Mutagenesis, Nucleic Acid Electrophoresis, Sequencing

    10) Product Images from "A low-cost open-source SNP genotyping platform for association mapping applications"

    Article Title: A low-cost open-source SNP genotyping platform for association mapping applications

    Journal: Genome Biology

    doi: 10.1186/gb-2005-6-12-r105

    Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.
    Figure Legend Snippet: Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.

    Techniques Used: Polymerase Chain Reaction, Ligation, Amplification, Sequencing, Labeling

    11) Product Images from "Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2?-deoxyadenosine"

    Article Title: Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2?-deoxyadenosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh542

    Incorporation of c 3 dATP by three Family A polymerases [Taq, Bst and Klenow (exo – )]. 20% polyacrylamide gel. Incubations used the SS primer (5′-GCGTAATACGACTCACTATAG-3′), the T Template (3′-CGCATTATGCTGAGTGATATCTGCGCAGAG-5′) and either dATP or c 3 dATP (indicated below lane) alone for 2 min or with dATP or c 3 dATP for 2 min, followed by addition of dCTP, dGTP and TTP and an additional 2 min incubation. Unextended primer is at position N; addition of dATP or c 3 ).
    Figure Legend Snippet: Incorporation of c 3 dATP by three Family A polymerases [Taq, Bst and Klenow (exo – )]. 20% polyacrylamide gel. Incubations used the SS primer (5′-GCGTAATACGACTCACTATAG-3′), the T Template (3′-CGCATTATGCTGAGTGATATCTGCGCAGAG-5′) and either dATP or c 3 dATP (indicated below lane) alone for 2 min or with dATP or c 3 dATP for 2 min, followed by addition of dCTP, dGTP and TTP and an additional 2 min incubation. Unextended primer is at position N; addition of dATP or c 3 ).

    Techniques Used: Incubation

    ( A ) Loss of primer upon incubation with dATP, dCTP, dGTP and TTP, trap and Family A polymerases. The extension of the primer by the first nucleotide, dCTP opposite dG, is observed least with Taq, more so with Bst and most with Klenow fragment (exo – ). A small loss of extended DNA is observed after the addition of each subsequent nucleotide. Due to the small amount of primer initially extended by 1 nt by Taq, very little full-length product is observed. ( B ) Loss of DNA primer extension after addition of each nucleotide with c3dATP, dCTP, dGTP and TTP. Again, extension of the primer by the first nucleotide, dGTP opposite dC, is observed least with Taq, more so with Bst and most with Klenow fragment (exo – ). The relative loss of extended DNA with each subsequent nucleotide addition does not differ between polymerases.
    Figure Legend Snippet: ( A ) Loss of primer upon incubation with dATP, dCTP, dGTP and TTP, trap and Family A polymerases. The extension of the primer by the first nucleotide, dCTP opposite dG, is observed least with Taq, more so with Bst and most with Klenow fragment (exo – ). A small loss of extended DNA is observed after the addition of each subsequent nucleotide. Due to the small amount of primer initially extended by 1 nt by Taq, very little full-length product is observed. ( B ) Loss of DNA primer extension after addition of each nucleotide with c3dATP, dCTP, dGTP and TTP. Again, extension of the primer by the first nucleotide, dGTP opposite dC, is observed least with Taq, more so with Bst and most with Klenow fragment (exo – ). The relative loss of extended DNA with each subsequent nucleotide addition does not differ between polymerases.

    Techniques Used: Incubation

    12) Product Images from "Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages"

    Article Title: Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm168

    ( a ) Extension of primers using the indicated diastereoisomers of dPTPαS with Taq and 9°N (modified) DNA polymerases for the times indicated. The positions of migration of the unextended primer, primer extended by 1 nt and the primer extended by 2 nt, are indicated by P(N), N + 1, N + 2, respectively. The amount of oligonucleotide loaded to mark the position where the primer runs (lane 1) was less than for other lanes in the gel; ( b ) Exo III digestion of the products of 2-min primer extension reactions using the indicated diastereomers (S or R) of alpha-thio-dPTP (from Figure 5 a). Each primer extension product was purified with a QIAquick column and digested with 20 Units of Exo III for the indicated times. The control shows the degradation of 5′- 32 P-labeled primer Z-SS-S19 (25mer) in a duplex with unlabeled Z-51-Temp, establishing that these are degraded by Exo III.
    Figure Legend Snippet: ( a ) Extension of primers using the indicated diastereoisomers of dPTPαS with Taq and 9°N (modified) DNA polymerases for the times indicated. The positions of migration of the unextended primer, primer extended by 1 nt and the primer extended by 2 nt, are indicated by P(N), N + 1, N + 2, respectively. The amount of oligonucleotide loaded to mark the position where the primer runs (lane 1) was less than for other lanes in the gel; ( b ) Exo III digestion of the products of 2-min primer extension reactions using the indicated diastereomers (S or R) of alpha-thio-dPTP (from Figure 5 a). Each primer extension product was purified with a QIAquick column and digested with 20 Units of Exo III for the indicated times. The control shows the degradation of 5′- 32 P-labeled primer Z-SS-S19 (25mer) in a duplex with unlabeled Z-51-Temp, establishing that these are degraded by Exo III.

    Techniques Used: Modification, Migration, Purification, Labeling

    13) Product Images from "Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'"

    Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

    Journal: Nucleic Acids Research

    doi:

    Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
    Figure Legend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

    Techniques Used: Amplification, Sequencing, Isolation, Polymerase Chain Reaction

    14) Product Images from "Optimised ligation of oligonucleotides by thermal ligases: comparison of Thermus scotoductus and Rhodothermus marinus DNA ligases to other thermophilic ligases"

    Article Title: Optimised ligation of oligonucleotides by thermal ligases: comparison of Thermus scotoductus and Rhodothermus marinus DNA ligases to other thermophilic ligases

    Journal: Nucleic Acids Research

    doi:

    Comparison of the rates of ligation of octanucleotides for four DNA ligases. ( A ) This is an image produced from radiolabelled ligation reactions after electrophoresis through a 15% polyacrylamide gel (see Materials and Methods). The left hand side of the gel depicts the control reactions lacking, from left to right, ligase, ssDNA template and octanucleotide library. The rate of the ligation reaction is determined by the length of the ladder and the intensity of each of the bands. This experiment used a complete octanucleotide library as substrate for each reaction (see text for details). The image depicts the results of varying the amount of each ligase, from left to right: Tth , 0.1, 0.3, 0.6, 1.3, 2.6, 5.1 and 6.3 pmol; Ts , 0.1, 0.3, 0.7, 1.3, 2.7, 5.4 and 8.0 pmol; Rm , 0.1, 0.2, 0.4, 0.8, 1.6, 3.1 and 4.6 pmol; Taq , 40.0 fmol and 0.1, 0.2, 0.4, 0.8 and 1.5 pmol. ( B ) The 2D graphic represents the polymers from (A) that were analysed. The intensity of each of the bands is expressed as a percentage of all the bands. Only the bands corresponding to oligonucleotide ligations were analysed.
    Figure Legend Snippet: Comparison of the rates of ligation of octanucleotides for four DNA ligases. ( A ) This is an image produced from radiolabelled ligation reactions after electrophoresis through a 15% polyacrylamide gel (see Materials and Methods). The left hand side of the gel depicts the control reactions lacking, from left to right, ligase, ssDNA template and octanucleotide library. The rate of the ligation reaction is determined by the length of the ladder and the intensity of each of the bands. This experiment used a complete octanucleotide library as substrate for each reaction (see text for details). The image depicts the results of varying the amount of each ligase, from left to right: Tth , 0.1, 0.3, 0.6, 1.3, 2.6, 5.1 and 6.3 pmol; Ts , 0.1, 0.3, 0.7, 1.3, 2.7, 5.4 and 8.0 pmol; Rm , 0.1, 0.2, 0.4, 0.8, 1.6, 3.1 and 4.6 pmol; Taq , 40.0 fmol and 0.1, 0.2, 0.4, 0.8 and 1.5 pmol. ( B ) The 2D graphic represents the polymers from (A) that were analysed. The intensity of each of the bands is expressed as a percentage of all the bands. Only the bands corresponding to oligonucleotide ligations were analysed.

    Techniques Used: Ligation, Produced, Electrophoresis

    15) Product Images from "Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'"

    Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

    Journal: Nucleic Acids Research

    doi:

    Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
    Figure Legend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

    Techniques Used: Amplification, Sequencing, Isolation, Polymerase Chain Reaction

    16) Product Images from "Determining the One, Two, Three, or Four Long and Short Loci of Human Complement C4 in a Major Histocompatibility Complex Haplotype Encoding C4A or C4B Proteins"

    Article Title: Determining the One, Two, Three, or Four Long and Short Loci of Human Complement C4 in a Major Histocompatibility Complex Haplotype Encoding C4A or C4B Proteins

    Journal: American Journal of Human Genetics

    doi:

    Defining the RCCX modular structure using different genomic RFLP Southern blot analyses. A, Restriction patterns of Taq I RFLP for the five selected individuals on simultaneous hybridization of probes specific for 3′ RP, CYP21, and 3′ TNX. B, Psh AI RFLP hybridized to a 3′ RP probe. C, Bam HI RFLP hybridized to a 3′ TNX probe. D, Phenotyping of C4A and C4B proteins by immunofixation of EDTA-blood plasma resolved by HVAGE. E, Immunoblot analysis of Rg1 and Ch1 antigenic determinants in plasma C4 proteins after HVAGE. F, Genomic Southern blot analysis of C4A and C4B genes by Psh AI-RFLP (using Probe D) and by Psh AI- Pvu II RFLP (using probe 22–25). G, HLA class I and class II alleles of the five subjects.
    Figure Legend Snippet: Defining the RCCX modular structure using different genomic RFLP Southern blot analyses. A, Restriction patterns of Taq I RFLP for the five selected individuals on simultaneous hybridization of probes specific for 3′ RP, CYP21, and 3′ TNX. B, Psh AI RFLP hybridized to a 3′ RP probe. C, Bam HI RFLP hybridized to a 3′ TNX probe. D, Phenotyping of C4A and C4B proteins by immunofixation of EDTA-blood plasma resolved by HVAGE. E, Immunoblot analysis of Rg1 and Ch1 antigenic determinants in plasma C4 proteins after HVAGE. F, Genomic Southern blot analysis of C4A and C4B genes by Psh AI-RFLP (using Probe D) and by Psh AI- Pvu II RFLP (using probe 22–25). G, HLA class I and class II alleles of the five subjects.

    Techniques Used: Southern Blot, Hybridization

    17) Product Images from "Enzymatic Cleavage of 3’-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis"

    Article Title: Enzymatic Cleavage of 3’-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-64541-z

    Incorporation of 3’-Aep-dCMP by commercially available A-family DNA polymerases (AF-DNAPs). ( A ) Top, the schematic representation of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu. Bottom, DNA fragment analysis of the primer (N) and the primer plus an incorporated 3’-Aep-dCMP by BF (N + 1). ( B ) Activities of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu, respectively. The primer-extension assays were performed as described in the Methods using 0.1, 0.2, 0.4, 0.8, 2, 4, 10, 20, or 40 μM of 3’-Aep-dCTP in the reaction.
    Figure Legend Snippet: Incorporation of 3’-Aep-dCMP by commercially available A-family DNA polymerases (AF-DNAPs). ( A ) Top, the schematic representation of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu. Bottom, DNA fragment analysis of the primer (N) and the primer plus an incorporated 3’-Aep-dCMP by BF (N + 1). ( B ) Activities of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu, respectively. The primer-extension assays were performed as described in the Methods using 0.1, 0.2, 0.4, 0.8, 2, 4, 10, 20, or 40 μM of 3’-Aep-dCTP in the reaction.

    Techniques Used:

    Related Articles

    Sequencing:

    Article Title: Methyltransferase-directed covalent coupling of fluorophores to DNA covalent coupling of fluorophores to DNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04229eClick here for additional data file.
    Article Snippet: The Ado-6-amine and Ado-6-azide cofactor analogues were synthesized as reported by Lukinavičius et al. .. Preparation of sequence-specifically modified DNA Plasmid DNA (pUC19, NEB) (50 ng μL–1 ), an AdoMet cofactor analogue (150 μM Ado-6-amine or Ado-6-azide), and M.TaqI DNA methyltransferase (0.1 mg ml–1 ) were incubated in CutSmart buffer (New England Biolabs) for 2 h at 60 °C. .. Subsequently, 1 μL of proteinase K was added and the reaction was incubated for 1 h at 55 °C.

    Modification:

    Article Title: Methyltransferase-directed covalent coupling of fluorophores to DNA covalent coupling of fluorophores to DNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04229eClick here for additional data file.
    Article Snippet: The Ado-6-amine and Ado-6-azide cofactor analogues were synthesized as reported by Lukinavičius et al. .. Preparation of sequence-specifically modified DNA Plasmid DNA (pUC19, NEB) (50 ng μL–1 ), an AdoMet cofactor analogue (150 μM Ado-6-amine or Ado-6-azide), and M.TaqI DNA methyltransferase (0.1 mg ml–1 ) were incubated in CutSmart buffer (New England Biolabs) for 2 h at 60 °C. .. Subsequently, 1 μL of proteinase K was added and the reaction was incubated for 1 h at 55 °C.

    Plasmid Preparation:

    Article Title: Methyltransferase-directed covalent coupling of fluorophores to DNA covalent coupling of fluorophores to DNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04229eClick here for additional data file.
    Article Snippet: The Ado-6-amine and Ado-6-azide cofactor analogues were synthesized as reported by Lukinavičius et al. .. Preparation of sequence-specifically modified DNA Plasmid DNA (pUC19, NEB) (50 ng μL–1 ), an AdoMet cofactor analogue (150 μM Ado-6-amine or Ado-6-azide), and M.TaqI DNA methyltransferase (0.1 mg ml–1 ) were incubated in CutSmart buffer (New England Biolabs) for 2 h at 60 °C. .. Subsequently, 1 μL of proteinase K was added and the reaction was incubated for 1 h at 55 °C.

    Incubation:

    Article Title: Methyltransferase-directed covalent coupling of fluorophores to DNA covalent coupling of fluorophores to DNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04229eClick here for additional data file.
    Article Snippet: The Ado-6-amine and Ado-6-azide cofactor analogues were synthesized as reported by Lukinavičius et al. .. Preparation of sequence-specifically modified DNA Plasmid DNA (pUC19, NEB) (50 ng μL–1 ), an AdoMet cofactor analogue (150 μM Ado-6-amine or Ado-6-azide), and M.TaqI DNA methyltransferase (0.1 mg ml–1 ) were incubated in CutSmart buffer (New England Biolabs) for 2 h at 60 °C. .. Subsequently, 1 μL of proteinase K was added and the reaction was incubated for 1 h at 55 °C.

    Article Title: DNA barcodes for rapid, whole genome, single-molecule analyses
    Article Snippet: In all, we expect this approach to find widespread application in the quantitative study of mixed genome samples and, particularly in studying the sequence context of genome-wide events and processes, such as replication or protein binding, that are not directly accessible with sequencing technologies. .. Labelling of genomic DNA A 200 μl solution containing 1× CutSmart Buffer (NEB), 10 μg genomic DNA, 0.9 μg TaqI DNA methyltransferase (M.TaqI) and 750 μM AdoHcy-azide ( ) was prepared and incubated at 50°C for 1 h. Subsequently, 5μl 18mg/ml proteinase K (NEB)/0.1% Triton X-100 (Sigma-Aldrich) was added and this was incubated at 50°C for 1 h, before purification by GenElute Bacterial Genomic DNA kit (Sigma-Aldrich). ..

    Purification:

    Article Title: DNA barcodes for rapid, whole genome, single-molecule analyses
    Article Snippet: In all, we expect this approach to find widespread application in the quantitative study of mixed genome samples and, particularly in studying the sequence context of genome-wide events and processes, such as replication or protein binding, that are not directly accessible with sequencing technologies. .. Labelling of genomic DNA A 200 μl solution containing 1× CutSmart Buffer (NEB), 10 μg genomic DNA, 0.9 μg TaqI DNA methyltransferase (M.TaqI) and 750 μM AdoHcy-azide ( ) was prepared and incubated at 50°C for 1 h. Subsequently, 5μl 18mg/ml proteinase K (NEB)/0.1% Triton X-100 (Sigma-Aldrich) was added and this was incubated at 50°C for 1 h, before purification by GenElute Bacterial Genomic DNA kit (Sigma-Aldrich). ..

    other:

    Article Title: Derivation and maintenance of mouse haploid embryonic stem cells
    Article Snippet: TaqI (methyltransferase enzyme; New England Biolabs, cat. no. M0219S).

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    New England Biolabs taq dna polymerases
    PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp <t>DNA</t> ladder; Lane 1–2: complete PEAR reactions containing <t>Taq</t> DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.
    Taq Dna Polymerases, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs taq polymerase
    Amplification results of three target genes using <t>Taq</t> polymerase. (A) Attempted amplification of the three <t>DNA</t> targets ( mpb83 , Mb012, and LMHCC_RS00060 ) sequences by Taq in the presence of DMSO using 3St, 2St and TD protocols; mpb83 and LMHCC_RS00060 successfully showed amplicons using the three protocols. Mb012 failed to show an amplicon. (B) Attempts of amplification of the three DNA target sequences in the absence of DMSO using 3St, 2St and TD protocols showing no amplicon. mpb83 with added enhancer in 3St PCR is used as a positive control. Mb0129 with no enhancer in 3St PCR is used as a negative control.
    Taq Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs crimson longamp taq polymerase
    Derivative dissociation plots produced by different reagents using one microliter of lysate produced by homogenizing one H. armigera leg with 24 H. zea legs in 625 μl of modified squish buffer. (A) Amplification reactions made with KAPA SYBR Fast Master Mix (purple) and NEB <t>LongAmp</t> <t>Taq</t> polymerase and buffer with 2.5 mM MgCl2 (Red). Peaks representing 18S rRNA control amplicon and species-specific amplicons of H. armigera and H. zea are present in all reactions, but melting temperatures were different in two reagents. (B) An enlarged view of the derivative dissociation plot produced by amplification reactions with NEB LongAmp Taq polymerase and buffer. Although variations in dissociation plots between samples were observed, the 3-peak dissociation plot was clearly identifiable.
    Crimson Longamp Taq Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs taq
    ( A ) Schematic showing the use of PspOMI digestion to evaluate the retention rate of Z : P pair during the PCR amplification of <t>DNA</t> containing a single Aegis nucleoside (see Table 4 for the sequence of oligonucleotides used). First, the Z -template (Temp-R-72- Z ) was amplified for 30 cycles in the presence of dNTPs, d Z TP and d P TP using <t>Taq</t> DNA polymerase. The final amplicon duplexes contained two kinds of products: one retaining the Z : P pair (product 2), the other misincorporating dC:dG pair (product 1). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 72-meroligonucleotides (full-length product, FLP) and all the oligonucleotides [including 72-merand 39-mer(digested fragment)] reflects the retention rate of Z : P pair in recognition sequence during PCR amplification. ( B ) Retention rates of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside with Taq DNA polymerase. The experiments were carried out according to the above schematic ( Figure 4 A). Lane 1 (control 1): PspOMI digestion of PCR product amplified by using the standard template (Temp-R-72-C). Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM). Lane 2 (control 2): misincorporation rate of PCR amplification of the standard template (Temp-R-72-C) in the presence of d Z TP and d P TP. Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM), d Z TP and d P TP (varying as indicated). Lanes 3–5: Retention rates of Z : P pair during the PCR amplification of Z -template (Temp-R-72- Z ). Final concentrations of PCR reaction mixture: dNTPs (200μM each), d Z TP and d P TP (varying as indicated). The concentration of the forward and reverse primers was fixed at 0.25 μM, while the concentration of templates were 250 pM (lane 3), 25 pM (lane 4) and 2.5 pM (lane 5), respectively.
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    PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Journal: PLoS ONE

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0008430

    Figure Lengend Snippet: PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Article Snippet: Alternatively, in order to remove BSA, Taq DNA polymerases and excess dNTPs, PEAR products were phenol extracted, ethanol precipitated, washed three times with 75% ethanol, dried and resuspended in ddH2 O, and double digested by the addition of 1 volume of cleavage mixture containing 1× NEBuffer 4, and 1.0 u/µl each of Hpy99I and PspGI.

    Techniques:

    Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Journal: PLoS ONE

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0008430

    Figure Lengend Snippet: Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Article Snippet: Alternatively, in order to remove BSA, Taq DNA polymerases and excess dNTPs, PEAR products were phenol extracted, ethanol precipitated, washed three times with 75% ethanol, dried and resuspended in ddH2 O, and double digested by the addition of 1 volume of cleavage mixture containing 1× NEBuffer 4, and 1.0 u/µl each of Hpy99I and PspGI.

    Techniques: Produced

    Amplification results of three target genes using Taq polymerase. (A) Attempted amplification of the three DNA targets ( mpb83 , Mb012, and LMHCC_RS00060 ) sequences by Taq in the presence of DMSO using 3St, 2St and TD protocols; mpb83 and LMHCC_RS00060 successfully showed amplicons using the three protocols. Mb012 failed to show an amplicon. (B) Attempts of amplification of the three DNA target sequences in the absence of DMSO using 3St, 2St and TD protocols showing no amplicon. mpb83 with added enhancer in 3St PCR is used as a positive control. Mb0129 with no enhancer in 3St PCR is used as a negative control.

    Journal: bioRxiv

    Article Title: PCR procedures to amplify GC-rich DNA sequences of Mycobacterium bovis

    doi: 10.1101/2020.02.18.953695

    Figure Lengend Snippet: Amplification results of three target genes using Taq polymerase. (A) Attempted amplification of the three DNA targets ( mpb83 , Mb012, and LMHCC_RS00060 ) sequences by Taq in the presence of DMSO using 3St, 2St and TD protocols; mpb83 and LMHCC_RS00060 successfully showed amplicons using the three protocols. Mb012 failed to show an amplicon. (B) Attempts of amplification of the three DNA target sequences in the absence of DMSO using 3St, 2St and TD protocols showing no amplicon. mpb83 with added enhancer in 3St PCR is used as a positive control. Mb0129 with no enhancer in 3St PCR is used as a negative control.

    Article Snippet: Unsuccessful attempts were faced with Taq polymerase, OneTaq DNA Polymerase (NEB, M0480S), Platinum™ Pfx DNA Polymerase (Invitrogen, 11708039), Expand Long Template PCR System (an enzyme mix that contains thermostable Taq DNA Polymerase and a thermostable DNA polymerase with proofreading activity, Roche, 11681834001).

    Techniques: Amplification, Polymerase Chain Reaction, Positive Control, Negative Control

    Derivative dissociation plots produced by different reagents using one microliter of lysate produced by homogenizing one H. armigera leg with 24 H. zea legs in 625 μl of modified squish buffer. (A) Amplification reactions made with KAPA SYBR Fast Master Mix (purple) and NEB LongAmp Taq polymerase and buffer with 2.5 mM MgCl2 (Red). Peaks representing 18S rRNA control amplicon and species-specific amplicons of H. armigera and H. zea are present in all reactions, but melting temperatures were different in two reagents. (B) An enlarged view of the derivative dissociation plot produced by amplification reactions with NEB LongAmp Taq polymerase and buffer. Although variations in dissociation plots between samples were observed, the 3-peak dissociation plot was clearly identifiable.

    Journal: Journal of Insect Science

    Article Title: Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

    doi: 10.1093/jisesa/iev137

    Figure Lengend Snippet: Derivative dissociation plots produced by different reagents using one microliter of lysate produced by homogenizing one H. armigera leg with 24 H. zea legs in 625 μl of modified squish buffer. (A) Amplification reactions made with KAPA SYBR Fast Master Mix (purple) and NEB LongAmp Taq polymerase and buffer with 2.5 mM MgCl2 (Red). Peaks representing 18S rRNA control amplicon and species-specific amplicons of H. armigera and H. zea are present in all reactions, but melting temperatures were different in two reagents. (B) An enlarged view of the derivative dissociation plot produced by amplification reactions with NEB LongAmp Taq polymerase and buffer. Although variations in dissociation plots between samples were observed, the 3-peak dissociation plot was clearly identifiable.

    Article Snippet: Initial tests of species-specific fragment amplification were carried out using Crimson LongAmp Taq polymerase with a 45 s initial denaturation step at 95°C followed by 35 cycles of 15 s denaturation at 95°C, 10 s annealing at 60°C, and 30 s extension at 72°C.

    Techniques: Produced, Modification, Amplification

    ( A ) Schematic showing the use of PspOMI digestion to evaluate the retention rate of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside (see Table 4 for the sequence of oligonucleotides used). First, the Z -template (Temp-R-72- Z ) was amplified for 30 cycles in the presence of dNTPs, d Z TP and d P TP using Taq DNA polymerase. The final amplicon duplexes contained two kinds of products: one retaining the Z : P pair (product 2), the other misincorporating dC:dG pair (product 1). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 72-meroligonucleotides (full-length product, FLP) and all the oligonucleotides [including 72-merand 39-mer(digested fragment)] reflects the retention rate of Z : P pair in recognition sequence during PCR amplification. ( B ) Retention rates of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside with Taq DNA polymerase. The experiments were carried out according to the above schematic ( Figure 4 A). Lane 1 (control 1): PspOMI digestion of PCR product amplified by using the standard template (Temp-R-72-C). Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM). Lane 2 (control 2): misincorporation rate of PCR amplification of the standard template (Temp-R-72-C) in the presence of d Z TP and d P TP. Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM), d Z TP and d P TP (varying as indicated). Lanes 3–5: Retention rates of Z : P pair during the PCR amplification of Z -template (Temp-R-72- Z ). Final concentrations of PCR reaction mixture: dNTPs (200μM each), d Z TP and d P TP (varying as indicated). The concentration of the forward and reverse primers was fixed at 0.25 μM, while the concentration of templates were 250 pM (lane 3), 25 pM (lane 4) and 2.5 pM (lane 5), respectively.

    Journal: Nucleic Acids Research

    Article Title: Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity

    doi: 10.1093/nar/gkq1274

    Figure Lengend Snippet: ( A ) Schematic showing the use of PspOMI digestion to evaluate the retention rate of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside (see Table 4 for the sequence of oligonucleotides used). First, the Z -template (Temp-R-72- Z ) was amplified for 30 cycles in the presence of dNTPs, d Z TP and d P TP using Taq DNA polymerase. The final amplicon duplexes contained two kinds of products: one retaining the Z : P pair (product 2), the other misincorporating dC:dG pair (product 1). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 72-meroligonucleotides (full-length product, FLP) and all the oligonucleotides [including 72-merand 39-mer(digested fragment)] reflects the retention rate of Z : P pair in recognition sequence during PCR amplification. ( B ) Retention rates of Z : P pair during the PCR amplification of DNA containing a single Aegis nucleoside with Taq DNA polymerase. The experiments were carried out according to the above schematic ( Figure 4 A). Lane 1 (control 1): PspOMI digestion of PCR product amplified by using the standard template (Temp-R-72-C). Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM). Lane 2 (control 2): misincorporation rate of PCR amplification of the standard template (Temp-R-72-C) in the presence of d Z TP and d P TP. Final concentrations of PCR reaction mixture: dNTPs (200 μM each), forward and reverse primers (0.25 μM each), template (250 pM), d Z TP and d P TP (varying as indicated). Lanes 3–5: Retention rates of Z : P pair during the PCR amplification of Z -template (Temp-R-72- Z ). Final concentrations of PCR reaction mixture: dNTPs (200μM each), d Z TP and d P TP (varying as indicated). The concentration of the forward and reverse primers was fixed at 0.25 μM, while the concentration of templates were 250 pM (lane 3), 25 pM (lane 4) and 2.5 pM (lane 5), respectively.

    Article Snippet: Deep Vent (exo− ), Deep Vent (exo+ ), Taq and Phusion DNA polymerases were purchased from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Labeling, Concentration Assay

    ( A ) Schematic showing the use of PspOMI digestion to evaluate the misincorporation rate of Z and (or) P pair during the PCR amplifications of the standard template (see Table 3 for the sequence of oligonucleotides used). First, the standard template (Tem-R-81) were amplified for 30–40 PCR cycles in the presence of dNTPs (200μM each), d Z TP (25μM) and (or) d P TP (25μM). The final amplicon duplexes contained two kinds of products: one retaining the dC:dG pair (product 1), the other misincorporating Z : P pair (product 2). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 81-mer oligonucleotides (full-length product, FLP) and all the oligonucleotides [including 81-mer and 42-mer oligonucleotides (digested fragment)] represents the misincorporation rate of Z and (or) P in recognition sequence during PCR amplification. ( B ) Misincorporation rates of PCR amplification of the standard template in the presence of the AEGIS components using Deep Vent (exo + and exo − ) DNA polymerases at indicated pH values. Four parallel PCRs were performed to amply the standard template ( Table 3 ) containing a recognition sequence (5′-GGGCCC-3′), followed by digestion with PspOMI for 16 h. The ratio between the amount of full-length product (FLP) and all the oligonucleotides indicate the misincorporation rate and shown on the figure. Lane 1: negative control PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each), followed by digestion with PspOMI. Lane 2: five-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each) and d Z TP (25 μM), followed by digestion with PspOMI. Lane 3: five-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each) and d P TP (25 μM), followed by digestion with PspOMI. Lane 4: six-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each), d Z TP (25 μM) and d P TP (25 μM), followed by digestion with PspOMI. ( C ) Misincorporation rates of PCR amplification of the standard template in the presence of d Z TP and (or) d P TP using Taq and Phusion DNA polymerases at indicated pH values. The reactions followed the same protocol as in Figure 3 B except for the polymerases.

    Journal: Nucleic Acids Research

    Article Title: Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity

    doi: 10.1093/nar/gkq1274

    Figure Lengend Snippet: ( A ) Schematic showing the use of PspOMI digestion to evaluate the misincorporation rate of Z and (or) P pair during the PCR amplifications of the standard template (see Table 3 for the sequence of oligonucleotides used). First, the standard template (Tem-R-81) were amplified for 30–40 PCR cycles in the presence of dNTPs (200μM each), d Z TP (25μM) and (or) d P TP (25μM). The final amplicon duplexes contained two kinds of products: one retaining the dC:dG pair (product 1), the other misincorporating Z : P pair (product 2). The product mixtures (including product 1 and 2) were then digested by PspOMI, and the ratio between the amount of radio-labeled 81-mer oligonucleotides (full-length product, FLP) and all the oligonucleotides [including 81-mer and 42-mer oligonucleotides (digested fragment)] represents the misincorporation rate of Z and (or) P in recognition sequence during PCR amplification. ( B ) Misincorporation rates of PCR amplification of the standard template in the presence of the AEGIS components using Deep Vent (exo + and exo − ) DNA polymerases at indicated pH values. Four parallel PCRs were performed to amply the standard template ( Table 3 ) containing a recognition sequence (5′-GGGCCC-3′), followed by digestion with PspOMI for 16 h. The ratio between the amount of full-length product (FLP) and all the oligonucleotides indicate the misincorporation rate and shown on the figure. Lane 1: negative control PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each), followed by digestion with PspOMI. Lane 2: five-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each) and d Z TP (25 μM), followed by digestion with PspOMI. Lane 3: five-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each) and d P TP (25 μM), followed by digestion with PspOMI. Lane 4: six-letter PCR amplification of the standard template (Tem-R-81) in the presence of dNTPs (200 μM each), d Z TP (25 μM) and d P TP (25 μM), followed by digestion with PspOMI. ( C ) Misincorporation rates of PCR amplification of the standard template in the presence of d Z TP and (or) d P TP using Taq and Phusion DNA polymerases at indicated pH values. The reactions followed the same protocol as in Figure 3 B except for the polymerases.

    Article Snippet: Deep Vent (exo− ), Deep Vent (exo+ ), Taq and Phusion DNA polymerases were purchased from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Sequencing, Transmission Electron Microscopy, Amplification, Labeling, Negative Control