Is Klenow necessary for RT-PCR? - (Oct/30/2008 )
Hello everyone,
I didn't get full marks for a question on an exam, and I'm a little confused. Can anyone help clarify this for me? I did a search of BioForum and came up with good information, but couldn't put it all together in a way that answers my question. For simple derivation of cDNA, is Klenow fragment necessary?
The exam question was: "What is cDNA, and how is it derived?"
I said that cDNA is copy DNA. RNA template, primers, reverse transcriptase, and dNTPs were required to produce cDNA. The RNA template was reverse-transcribed 3-5' to form an RNA-DNA hybrid, which was then dissociated and the cDNA single strand became the template for the dsDNA product.
I dropped points for failing to address: 1) digesting with RNase H, 2) nick translation, 3) end-filling with terminal transferase, and 4) to use a Klenow fragment. I know why I missed the first three, but not the last. I have looked through my notes, and even re-read the passage in the book that my notes came from, but can't find any mention of Klenow in RT-PCR. In fact, Klenow isn't even mentioned until 600 pages after the section on RT-PCR. (He must have mentioned it in lecture.)
Can anyone else me please?
Thanks,
Stasha
I didn't get full marks for a question on an exam, and I'm a little confused. Can anyone help clarify this for me? I did a search of BioForum and came up with good information, but couldn't put it all together in a way that answers my question. For simple derivation of cDNA, is Klenow fragment necessary?
The exam question was: "What is cDNA, and how is it derived?"
I said that cDNA is copy DNA. RNA template, primers, reverse transcriptase, and dNTPs were required to produce cDNA. The RNA template was reverse-transcribed 3-5' to form an RNA-DNA hybrid, which was then dissociated and the cDNA single strand became the template for the dsDNA product.
I dropped points for failing to address: 1) digesting with RNase H, 2) nick translation, 3) end-filling with terminal transferase, and 4) to use a Klenow fragment. I know why I missed the first three, but not the last. I have looked through my notes, and even re-read the passage in the book that my notes came from, but can't find any mention of Klenow in RT-PCR. In fact, Klenow isn't even mentioned until 600 pages after the section on RT-PCR. (He must have mentioned it in lecture.)
Can anyone else me please?
Thanks,
Stasha
the klenow fragment is a DNA polymerase that has its 5' → 3' exonuclease activity part cleaved of.
My guess is that your lecturer wanted you to describe why an enzyme with a 5' → 3' exonuclease activity is unsuited for the synthesis of double-stranded DNA from single-stranded templates.
btw, cdna stands for "complementary" dna (complementary to the mrna used as template).
see the wikipedia article:
cDNA
see the wikipedia article:
cDNA
Actually, Weaver (our text) lists both as acceptable terms.