second strand cDNA synth without primer? - (Feb/12/2008 )

Howdy all,

I'm generating cDNA from purified poly(A)RNA, following a protocol and noticed that there is no second strand primer. AND, the thermocycling conditions for the second strand are fairly cold - 2hrs @ 16C. Is it possible to have cDNA synthesis occur spontaneously without a primer? I'm wondering if this is a missing element in my protocol; the first strand primer (a polyT primer) shouldn't match the first strand synthesized, so what starts the second strand??? And what does the DTT do?

Thanks for any info!

Here's the scheme - most reagents are supplied by Invitrogen:

First Strand Synth -
start with:
polyA RNA
dT (polyT) primer
dNTPs
water

5min@65C, 2min@4C, next add:
first strand buffer
DTT (what the heck is that?? comes with the superscript kit)
RNAseOUT
SuperScript III RT

1hr@50C, 15min@70C, 2min@4C, next add:

Second Strand Synth
second strand buffer
dNTP
DNA ligase
DNA polymerase
RNase H
water

2hr@16C, next add:
T4 DNA polymerase

5min@16C, next add:
EDTA to stop rxn.

DTT stands for dithiothreitol, a reducing agent. It probably is used to prevent the reverse transcriptase from oxidative damage.

From the looks of thing your cDNA synthesis method uses RNA replacement to synthesis the second strand. Basically after the reverse transcription (first strand synthesis), you have a RNA-DNA duplex. Treatment with the RNAse H during the second step, cuts up the RNA, leaving small fragment bound to the DNA strand. These bits of of RNA serves as primers for the synthesis of the second DNA strand.

FYI- ssDNA can loop back on itself froming a hair pin loop. The free end can thus act as a primer for the synthesis of the second DNA strand. However leaving things to chance is not the best of things.

ah, brilliant!

i like it so much more when i understand things. Thanks!!