What is inverse PCR?

[Yoonsoo Hahn]

In article <33CE2A30.16A9 at Bristol.ac.Uk>, Harry Witchel
<Harry.Witchel at Bristol.ac.Uk> wrote:

> Hello Molecular Deities --
>         What is inverse PCR?  I can't find a definition anywhere.
> Thanks,
> Harry.Witchel at Bristol.ac.uk

You can amplify the flanking regions of the known sequence by Inverse
PCR (I-PCR). Primers are designed opposite orientation, so it's called
"Inverse" PCR.
First, DNA is digested by restriction enzyme, and then ligated to
generate circularized fragment. I-PCR is a good method for rapid
cloning of flanking sequences.

Okay... Here's a schematic diagram.

(1) Restriction Digestion

    unknown 5' flanking     known sequence     unknown 3' flanking
    |---------------------===================---------------------|
                            <--        -->
                          primer 1     primer 2

(2) Ligation to generate circularized molecule.

               primer 1   primer 2
                <--       -->
        ------==================-----
       (                             )
        -------------|---------------
           fused unknown flanking regions

(3) PCR product

      primer 1                            primer 2
      -->                                      <--
      =====----------------|-----------------=====
              fused unknown flanking regions

Got it?

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