Is cDNA a single or double stranded ? - (Feb/09/2006 )

Hello,

I posted my question elsewhere but hadn't answer, I'd like to post it here and, sorry it seems stupid question, because I'm really confused !

I read in somwhere a thing which perplexed me about cDNA. I'd like to know if cDNA is a double stranded or a single stranded ?

I know that cDNA is the copy of mRNA by reverse transcriptase so, theorically, it would be a single stranded , right or I'm mistake ?

Thanks for your feedback

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For most cases, such for RT-PCR, 3' or 5' RACE, we just use single stranded cDNA (the first strand cDNA), while in case of preparing the cDNA for library construction, or RDA/SSH analysis which might need additional second strand synthesis step for generate double strand cDNA.

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hi
you need to differenciate 2 things :

cDNA in general is the DNA molecule corresponding roughly to the mRNA seuqence. So It's a double stranded molecule included in plasmid for expression in most cases.

RT PCR : the mRNA molecule is reverse transcribed in cDNA single strand. After this point RNase eliminates the mRNA molecule and you get a single stranded cDNA molecule. As the next step is quite classical PCR, a single strand is ok for the exp (and the first step, which corresponds to sysnthesis of the corrseponding second strand of a DNA molecule restores the real cDNA molecule.

To be right, cDNA is a double stranded molecule, but for convenience, cDNA is also used for designing the reverse transcribed molecule of the RTPCR. It should be named as half cDNA or single strand cDNA.
cDNA is a shorting name.

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while in most cases, cDNA, abbreviate of complementary DNA, is definited as a single-strand DNA molecule with a nucleotide sequence that is complementary to an RNA molecule, and is synthesised in the lab from mRNA by reverse transcription...

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So, cDNA is single stranded until a PCR reaction is carried out ? Ok, in this case, how Taq DNA Polymerase generates a second complementary strand from only one ? When the two primers anneal to each side of the same strand, how they can produce the second strand (the new complementary one) ?

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In PCR one primer binds to one single stranded piece of DNA (that is why the first step is always to dentature at 94C(or so) to seperate any double stranded molecules into single strands. In the case of the first-strand cDNA from an RT reaction only one primer binds in the first round... This means that the first round of amplification in an RT-PCR is not logrithmic you simply synthesize the second strand of cDNA. In subsequent rounds (round 2 to round n) there is logrithmic amplification because both the forward and reverse primers bind to the denatured double stranded template.

In the first round, I think it is the forward primer that binds... I think this is because mRNA=forward strand in the genome and the first strand cDNA=complement of mRNA (or is the same as the reverse strand in the genome)

Someone check my logic here, I am confusing myself... is that right that the mRNA is the complement of the reverse strand of genomic DNA??
ie: mRNA sequence=forward strand=5'-3' sequence in gDNA (just with U not T)???

HTH

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Thank you for all, It's more clear for me now.

I think so, yes, until an intron is encountered, right ?
We can check any of your known mRNA sequences in NCBI, recover the genomic hit and compare the sequences to see if it's true or not.

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Yup that's right, you'd have to skip the introns

Sounds like a good way to test will try later if I get a chance and let you know... the more I think about it, mRNA sequence=forward strand sequence has to be right though...

Thanks!

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