Journal: Nucleic Acids Research
Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase
Figure Lengend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.
Article Snippet: For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).
Techniques: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification