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PCR of unknown DNA


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#1 nystoney

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Posted 15 November 2004 - 08:14 AM

I want to PCR amplify DNA (the sequence of which may not be known) by ligating a known linker to both ends of the DNA sample and PCRing with same linkers (oligos). I'm familiar with Clontech's Marathon cDNA Amplification Kit (cat no K1802) for doing this, but was wondering if anyone knows of other suppliers with such kits? Is this a commonly used approach in molecular biology?

#2 Dr.B

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Posted 24 November 2004 - 02:02 AM

This is a commonly used approach, e.g. in RDA (Lisitsyn et al 1993), and there are improvements called vectorette, sprinklerette etc.
you can order oligonucleotides and use them as linkers
Important: do you use a digest which you want to amplify? Then use compatible linkers. Note that oligonucleotides normally are not 5'phosphorylated, this is in fact an advantage.
If you want to ligate linkers to blunt-end DNA, make sure you have a double-stranded linker.
good luck

#3 DaveC

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Posted 25 November 2004 - 08:29 AM

and there are improvements called vectorette, sprinklerette etc.

Vectorette primers seem like such a clever idea, yet I can find so few papers where they are used. Is there a reason why it hasn't taken off?




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