Journal: PLoS ONE

Article Title: Platinum nanoparticles induce damage to DNA and inhibit DNA replication

doi: 10.1371/journal.pone.0180798

Figure Lengend Snippet: (A) The suggested effects of PtNPs on polymerase chain reaction (PCR) is based on binding of PtNPs to the Taq DNA polymerase, which leads to ceasing of PCR, (B) whereas CisPt primarily intercalates in DNA structure and stops PCR by this way. The gel electrophoregrams of PCR product mixture with particular concentration of (C) PtNPs (0.04–4 200 ng/mL of Pt) and (D) CisPt (0.04–42 000 ng/mL of Pt). (E) DNA denaturation temperature affected by the 0–200 μg/mL of Pt derivatives. Fluorescence of labelled nucleotides of DNA fragment after sequencing, which was influenced by (F) 0–20 μg/mL of PtNPs and (G) 0–0.33 μg/mL of CisPt. For all measurement n = 3.

Article Snippet: The volume of the reaction mixture was 25 μL, which was composed of 2.5 μL of 10× standard Taq reaction buffer, 0.5 μL of 1 mM deoxynucleotide solution, 0.5 μL of each of the primers (10 μM), 0.125 μL of Taq DNA polymerase; selected volume of water or drugs diluted with water (sterile, ACS purity, Sigma-Aldrich) and 0.5 μL of bacteriophage λ DNA.

Techniques: Polymerase Chain Reaction, Binding Assay, Concentration Assay, Fluorescence, Sequencing

Journal: Medicine

Article Title: Sox17 Promoter Methylation in Plasma DNA Is Associated With Poor Survival and Can Be Used as a Prognostic Factor in Breast Cancer

doi: 10.1097/MD.0000000000000637

Figure Lengend Snippet: Methylation status of the Sox17 promoter in breast cancer tissues and paired plasma DNA. (A) Representative results of MSP assays of Sox17 methylation in primary breast cancer tissues; B. Representative results of MSP of Sox17 methylation in paired plasma DNA. DW = distilled water, M = methylated, MSP = methylation-specific polymerase chain reaction, NC = negative control, P = plasma DNA, PC = positive control, T = breast cancer tissue, U = unmethylated.

Article Snippet: Each MSP reaction included 2 μL of DNA template, 0.18 μL of each primer, 0.45 μL of 10 mM dNTP Mix (Promega Corp., Madison, WI, USA) 1.5 μL 10× PCR buffer, and 0.12 μL of HotStart Taq DNA Polymerase (Sigma, Germany) in a final reaction volume of 15 μL.

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

Journal: Nucleic Acids Research

Article Title: Mechanical properties of DNA-like polymers

doi: 10.1093/nar/gkt808

Figure Lengend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

Article Snippet: For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara).

Techniques: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

Journal: BMC Biochemistry

Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

doi: 10.1186/1471-2091-12-47

Figure Lengend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

Article Snippet: BrdUTP and dNTPs were from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Incubation, Labeling

Journal: PLoS Pathogens

Article Title: Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape

doi: 10.1371/journal.ppat.1002721

Figure Lengend Snippet: Adsorption of plasma Nabs by autologous T/F Env monomers and trimers. Plasma from CH40 (A), CH77 (B), and CH58 (C) was incubated with magnetic-bead bound gp120 or tethered gp140 protein corresponding to the T/F sequence from each subject. Beads were removed and neutralization assessed by TZM-bl assay (BSA, bovine serum albumen; b12 broadly neutralizing mAb positive control). Results are the mean +/− SD of three independently performed experiments each performed in duplicate.

Article Snippet: In-gel PCR amplification was then performed with a 300 µl PCR solution (1 µM primer (CH40/77-for), 0.1% Tween-20, 0.2% BSA, 1× PCR buffer, 100 µM dNTP mix and 3.3 units of Jumpstart Taq DNA polymerase (Sigma, St. Louis, MO) under a sealed SecureSeal chamber (Grace Bio-Labs, Inc., Bend, OR) in a PTC-200 Thermal Cycler.

Techniques: Adsorption, Incubation, Sequencing, Neutralization, Positive Control

Journal: British Journal of Cancer

Article Title: Interleukin-8/CXCL8 is a growth factor for human lung cancer cells

doi: 10.1038/sj.bjc.6602227

Figure Lengend Snippet: Expression of IL-8 mRNA and protein in lung cancer cell lines. Expression of IL-8 mRNA was measured by RT–PCR ( A ). A 1 μ g portion of total RNA was reverse-transcribed for PCR reactions of IL-8 and control GAPDH. The expected 289 bp band of IL-8 mRNA was strongly expressed in A549, H460 and MOR/P, but was undetectable in all SCLC cell lines. Production of IL-8 protein was measured by ELISA ( B ). Conditioned medium was collected after 1 × 10 6 cells were cultured in serum-free RPMI medium for 48 h. Each bar is the mean±s.e. of three determinations from two independent experiments.

Article Snippet: A 0.5 μ g portion of total RNA was reverse-transcribed for subsequent PCR amplification for each pair of primers in a volume of 10 μ l, including 10 U of enhanced AMV reverse transcriptase (Sigma, Poole, Dorset, UK), 20 U of RNase inhibitor (Sigma), 0.5 μ g of oligo(dT)15 primer, 0.5 nmol of each dNTP and 1 × first-strand buffer (50 mM Tris-HCl, pH 8.3, 40 mM KCl, 8 mM MgCl2 , 1 mM dithiothreitol) provided by Sigma.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: British Journal of Cancer

Article Title: Interleukin-8/CXCL8 is a growth factor for human lung cancer cells

doi: 10.1038/sj.bjc.6602227

Figure Lengend Snippet: Expression of CXCR1 and CXCR2 in lung cancer cell lines. Expression of CXCR1 and CXCR2 proteins on the cell surface was measured by flow cytometry with mouse anti-human CXCR1 (R1) and CXCR2 (R2) and mouse IgG as control (C). Representative flow cytometric histograms of A549 and Lu165 showing the low expressions of CXCR1 and CXCR2 in A549 ( A ) and high expressions of CXCR1 and CXCR2 in Lu165 ( B ), respectively. Percentage of positive cells of CXCR1 and CXCR2 in nine lung cancer cell lines are summarised in ( C ). Each bar is the mean±s.e. of four independent experiments. Expression of CXCR1 and CXCR2 mRNA was measured by RT–PCR in A549 and H460 (as a positive control) ( D ). Total RNA (1.5 μ g) was reverse-transcribed for PCR reactions of CXCR1, CXCR2 and control GAPDH. The expected 512 bp band of CXCR1 was expressed in A549 and H460. The expected 202 bp band of CXCR2 was expressed in control cell H460 but not in A549 cells.

Article Snippet: A 0.5 μ g portion of total RNA was reverse-transcribed for subsequent PCR amplification for each pair of primers in a volume of 10 μ l, including 10 U of enhanced AMV reverse transcriptase (Sigma, Poole, Dorset, UK), 20 U of RNase inhibitor (Sigma), 0.5 μ g of oligo(dT)15 primer, 0.5 nmol of each dNTP and 1 × first-strand buffer (50 mM Tris-HCl, pH 8.3, 40 mM KCl, 8 mM MgCl2 , 1 mM dithiothreitol) provided by Sigma.

Techniques: Expressing, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Mechanical properties of DNA-like polymers

doi: 10.1093/nar/gkt808

Figure Lengend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

Article Snippet: For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara).

Techniques: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

Journal: Journal of Applied Genetics

Article Title: Molecular screening for avirulence alleles AvrLm1 and AvrLm6 in airborne inoculum of Leptosphaeria maculans and winter oilseed rape (Brassica napus) plants from Poland and the UK

doi: 10.1007/s13353-014-0235-8

Figure Lengend Snippet: Seasonal fluctuations in DNA quantities or ascospore counts obtained by sampling airborne propagules collected over two autumns ( a , b ; 2002 and c , d ; 2006) periods on Melinex tapes of a Burkard 7-day volumetric air sampler located in Rothamsted Research, Harpenden, UK. Quantitative PCR assays detected avirulence alleles AvrLm1 ( a , c ; black line ), AvrLm6 ( a , c ; grey line ) or with primers based on β -tubulin ( b , d ; black line ) or e rg11 ( b , d ; grey line ) fragments in the propagules of Leptosphaeria maculans . Ascospore release patterns ( b , d ; dotted line ) were determined by light microscopic counts of Leptosphaeria -like ascospores per m 3 of air

Article Snippet: Quantitative PCR to assess proportions of DNA of each Leptosphaeria maculans in the spore samples For quantitative PCR, a standard 20-μL reaction contained 5 μL template DNA, 200 nM forward primer, 180 nM reverse primer (Mahuku et al. ; Liu et al. ), 10 μL SYBR Green JumpStart Taq ReadyMix (Sigma, UK), 0.08 μL ROX internal reference dye (Sigma, UK) and 3.78 μL nuclease-free, sterile water.

Techniques: Sampling, Real-time Polymerase Chain Reaction

Journal: Journal of Applied Genetics

Article Title: Molecular screening for avirulence alleles AvrLm1 and AvrLm6 in airborne inoculum of Leptosphaeria maculans and winter oilseed rape (Brassica napus) plants from Poland and the UK

doi: 10.1007/s13353-014-0235-8

Figure Lengend Snippet: Seasonal fluctuations in DNA quantities or ascospore counts obtained by sampling airborne propagules collected over two autumns ( a , b ; 2006 and c , d ; 2008) periods on Melinex tapes of a Burkard 7-day volumetric air sampler located in Poznan, Poland. Quantitative PCR assays detected avirulence alleles AvrLm1 ( a , c ; black line ), AvrLm6 ( a , c ; grey line ) or with primers based on β -tubulin ( b , d ; black line ) or e rg11 ( b , d ; grey line ) fragments in the propagules of Leptosphaeria maculans . Ascospore release patterns ( b , d ; dotted line ) were determined by light microscopic counts of Leptosphaeria -like ascospores per m 3 of air

Article Snippet: Quantitative PCR to assess proportions of DNA of each Leptosphaeria maculans in the spore samples For quantitative PCR, a standard 20-μL reaction contained 5 μL template DNA, 200 nM forward primer, 180 nM reverse primer (Mahuku et al. ; Liu et al. ), 10 μL SYBR Green JumpStart Taq ReadyMix (Sigma, UK), 0.08 μL ROX internal reference dye (Sigma, UK) and 3.78 μL nuclease-free, sterile water.

Techniques: Sampling, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART)

doi: 10.1038/s41598-018-36207-4

Figure Lengend Snippet: Copy number determination using quantitative PCR against standard curve, Quant Studio 3D digital PCR and TapeStation analysis. ( a ) Quantitation of 0.1 percent Certified Reference Material (CRM) Bt11 DNA extract and known dilutions of linearised plasmid pART7[49] containing the 35Sp sequence incrementally from 500000 copies (pink) to 5 copies per 5 microlitres (dark green). Dilutions of the 0.1 percent Bt11 extract were; mid-green for undiluted, mid-blue for 1:2 dilution and light brown for 1:5 dilution. NTCs: pale green. ( b ) Derived standard curve using pART7 data. Standard curve of this linear plasmid DNA has calculated PCR efficiency of 101.3 percent with R2 = 0.9987. ( c ) Melt curve analysis of amplicons from ( a ). ( d ) TapeStation analysis of genomic DNA for undiluted 0.1 percent Bt11 and 5.0 percent NK603 CRM DNA extracts using an Agilent genomic DNA ScreenTape. ( e ) Quant Studio 3D digital PCR results using 35Sp primers with a TaqMan probe (see Methods) showing negative partitions in yellow and positive partitions in blue from which the concentration of the samples are calculated (Table 1 ).

Article Snippet: The reaction volume of 20 microlitres consisted of 5 microlitres of extracted DNA template or non-template control with molecular grade water, 10 microlitres of JumpStart, 5 picomoles of each respective primer (Sigma HPLC purified targeting the CaMV 35S promoter and the NOS terminator sequences) made up to volume with molecular grade water.

Techniques: Real-time Polymerase Chain Reaction, Digital PCR, Quantitation Assay, Plasmid Preparation, Sequencing, Derivative Assay, Polymerase Chain Reaction, Concentration Assay

Journal: Scientific Reports

Article Title: The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

doi: 10.1038/srep14525

Figure Lengend Snippet: mRNA expression levels of PvACR2 and Pv2.5–8 in P. vittata fronds (a), roots (b) and gametophytes (c) by qRT-PCR. Green columns indicate controls whereas brown columns indicate As treatment. Each value represents the mean of three qRT-PCR replicates (n = 3) and its standard errors (±SE) repeated at least three times for each cDNA, from three different RNA extractions. Values followed by the same letter are not different, according to Fisher’s least significant difference test at a p ≤ 0.05.

Article Snippet: 1–2 μg of total RNA from P. vittata tissues were treated with Dnase I (Sigma-Aldrich) and reverse transcripted to cDNAs using oligo (dT)18 primer and RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, Canada).

Techniques: Expressing, Quantitative RT-PCR

Journal:

Article Title: Chromosome-specific DNA Repeat Probes

doi: 10.1369/jhc.6A6974.2006

Figure Lengend Snippet: (A) Agarose gel showing an inverted image of PCR products and sizemarker DNAs after ethidium bromide staining. Lane L1: chromosome 18 PCR, genomic DNA as template; L2: chromosome 17 PCR, genomic DNA as template; L3: chromosome 17 PCR, BAC RP11-285M22

Article Snippet: For PCR, 100 ng genomic DNA (Sigma) or BAC DNA was used as amplification templates.

Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Staining, BAC Assay

Journal: Applied and Environmental Microbiology

Article Title: Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum

doi: 10.1128/AEM.71.9.5511-5522.2005

Figure Lengend Snippet: Linear view of consensus optical maps with DNA sequence-based maps. Solid black arrows represent the locations of missing cuts in the consensus optical maps. The XbaI and NheI consensus optical maps extend to the left of the origin of the DNA sequence-based map because both optical maps had a missing cut at that position.

Article Snippet: Surface properties were assayed by digesting lambda DASH II bacteriophage DNA with 40 units of XbaI, HindIII, and NheI diluted in 200 μl of digestion buffer with 0.2% Triton X-100 (Sigma, St. Louis, MO) at 37°C to determine optimal digestion times, which ranged from 30 to 120 min.

Techniques: Sequencing

Journal: Applied and Environmental Microbiology

Article Title: Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum

doi: 10.1128/AEM.71.9.5511-5522.2005

Figure Lengend Snippet: Whole-genome circular XbaI map of R. rubrum . The outermost circle (thick line) represents the consensus map created by Gentig from the single-molecule maps shown as arcs. The single-molecule maps were made from single DNA molecules digested with XbaI. The color ordering of the fragments is for contrast and is therefore random.

Article Snippet: Surface properties were assayed by digesting lambda DASH II bacteriophage DNA with 40 units of XbaI, HindIII, and NheI diluted in 200 μl of digestion buffer with 0.2% Triton X-100 (Sigma, St. Louis, MO) at 37°C to determine optimal digestion times, which ranged from 30 to 120 min.

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum

doi: 10.1128/AEM.71.9.5511-5522.2005

Figure Lengend Snippet: Comparisons of the XbaI, NheI, and HindIII optical maps to sequence data. (A, B, and C) Plots of optical map fragment sizes versus the DNA sequence-based map fragment sizes from the finished sequence for XbaI (A), NheI (B), and HindIII (C). The error bars represent the standard deviation of optical map fragment size about the mean. (D, E, F) Plots of the relative fragment sizing error [100 × (optical map fragment size - corresponding DNA sequence-based map fragment size)/corresponding DNA sequence-based map fragment size] versus DNA sequence-based map fragment size for XbaI ( D), NheI (E), and HindIII (F).

Article Snippet: Surface properties were assayed by digesting lambda DASH II bacteriophage DNA with 40 units of XbaI, HindIII, and NheI diluted in 200 μl of digestion buffer with 0.2% Triton X-100 (Sigma, St. Louis, MO) at 37°C to determine optimal digestion times, which ranged from 30 to 120 min.

Techniques: Sequencing, Standard Deviation

Journal: PLoS ONE

Article Title: Direct Chromatin PCR (DC-PCR): Hypotonic Conditions Allow Differentiation of Chromatin States during Thermal Cycling

doi: 10.1371/journal.pone.0044690

Figure Lengend Snippet: Optimization of DC-PCR for drug screening. Effect of cytoplasmic enzymes on DC-PCR ( A ). KMS-12-PE cells were diluted at 1∶25 in hypotonic PCR buffer with or without added protease inhibitors and incubated for 20 min before PCR was run to simulate the time required for pipetting during a small molecule screen. Inclusion of protease inhibitors in the PCR mastermix decreased the number of amplifiable sites in DAC treated and untreated KMS-12-PE cells suggesting that digestion of DNA binding proteins can occur in PCR buffer although epigenetic differences between samples are maintained in the studied example. Detection of DC-PCR product via UV transillumination of PCR plates and sensitivity compared to gel electrophoresis ( B ). Ethidium bromide was added to the PCR mastermix before serially diluted cells were submitted to DC-PCR: untreated, DAC treated or fibroblasts. After completion of thermal cycling samples were first analyzed under UV light (Left panel), then via agarose-gel electrophoresis (Right panel). Using gel-based detection, the level of detection for epigenetic differences of CDKN2A was lower, down to about 1 cell for site #6 compared to about 300 cells with direct UV transillumination. The primer numbers correspond to sequences on the map described in Figure 1A . Three independent experiments yielded the same detection levels. Correlation between direct UV transillumination and gel electrophoresis was seen in all experiments (hundreds).

Article Snippet: PCR mix composition: 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2 , 200 µM dNTP, 1 U Taq-polymerase (New England Biolabs Inc.), 0.2 µM of corresponding forward and reverse primers, pH 8.3, supplemented with 1x Proteinase inhibitor cocktail, Set #5 (EMD Chemicals, Inc.) and 1 µM ethidium bromide (Sigma-Aldrich Co.

Techniques: Polymerase Chain Reaction, Incubation, DNA Binding Assay, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis