How was the 2nd strand synthesized with only DNA poly and RNase H?

[woshiliangliang]

Hi guys,

When prerparing an illumina RNA-seq library, i found this instruction:

In secondary strand synthesis the DNA polymerase use the first-stranded cDNA as template and the RNase H digested mRNA as primers to synthesize DNA. Some where the 5' RNA could be replaced and the fragments of double stranded cDNA could be ligated by the E.coli DNA ligase in the reaction.

I believe there's no DNA ligase added in this reaction for my library, meanwhile i dont think DNA poly has ligase activity. So i'll appreciate anyone who helps me understand better about how this works.

Thanks.

[luc]

Hi,
some 2nd strand synthesis kits for library prep include E.coli ligase (the NEB kit for example), but most don't, as do the protocols that I am familiar with. I don't know about Illumina - they usually do not give away the details.
In short, ligation seems not necessary. Likley DNA Pol.1 does a good enough job at strand displacement for short fragments.