Scheme of fragment library preparation
Материал из Zbio
Protocols are quite similar.
[править] DNA digestion
performed by ultrasound treatment or by hydrodinamic forces.
[править] End-repair reaction
Three enzymes work simultaneously:
- T4 DNA polymerase: digestion of 3' protruding ends;
- Klenow DNA polymerase: extension of 3' recessive ends;
- T4 PNK: phosphorilation of 5' ends & dephosphorilation of 3' ends, the best substrates are: protruding and blunt ends.
In Illumina protocol prevents self ligation and ligation of one fragment to another. In spite A-tailing reaction has a preference to some end-nucleotides, ligation is quantitative (e.g. all end-nucleotides are tailed and all end are ligated to adaptors).
[править] Adaptor ligation
one Y-shape adapter is used. One oligonucleotide of Y-adapter ligates to 5'-ends of all DNA molecules, another oligonucleotide — to 3'-ends. Advantages of such approach:
- 100% of DNA fragments acquire two diffrent oligonucleotides on 5' and 3' ends;
- definite orientation of adaptors;
- Y-adapter is phosphorilated, so nick-translation is not necessary.
Y-adaptor has 3' T-protruding nucleotide on the double-stranded stem. It prevents self-ligation of adaptors. A single-stranded 3'-end of Y-adaptor is blocked for extension and ligation to prevent background during ligation and PCR.
two different double-stranded adaptors are used. For combinatorial reasons only 1/2 of molecules would have different adaptors on the ends. Contrary to Illumina approach, adaptor may be in both orientations. Blunt-ends of adaptors are dephosphorilated to prevent adaptor-adaptor ligation.
[править] Nick translation
have to be done in SOLiD protocol because: adaptors was dephosphorilated, so they covalently attache only to 5'-ends of DNA fragments. Nick-translation can't be performed on PCR-stage with a current protocol, because chemically inactivated polymerase is used (fragment denaturated before nick-translation). With antibody-inactivated enzyme nick-translation may be combined with PCR.
removes adaptor dimers. Fragments with similar length gives more uniform clonal amplification.
increases the number of library molecules. On the clonal amplification step not each molecule is converted to the cluster (Illumina) or bead (SOLiD). The problem, related with preamplification is, that the same molecule may be sequenced several times (this reduces efficiency of sequencing).
PCR-1 primer is shorter than adaptor. DNA fragments with P1-P1 adaptors do not amplify because of suppression of PCR by panhandle structure. DNA fragments with P2-P2 adaptors amplified, but they have no chance to stick to paramagnetic beads during ePCR. SOLiD protocol is relatively sensitive to overamplification, because library is loaded in emulsion in double-stranded form.
Preamplification is obligatory, because one of the bridge-amplification sites is introduced during preamplification (Y-adaptor is shorter, than one of the PCR-primers). Illumina protocol is relatively unsensitive to overamplification, because library is loaded on flowcell in a single-stranded form.