On 20 Sep 1999 07:20:53 -0700, huangwei at LIFE.SDU.EDU.CN (huangwei)
wrote:
> I am a postgraduate of Shangdong University in China.Recently,I am engaged in the
>work concerning the technology of inverse PCR.I eagerly want to know how can I
>manipulate IPCR successfully and what the key is.
> Thanks a lot!
there are three keys to inverse PCR:
1) try many 6-base cutters. more frequent cutters yield very short
flanking sequences. any one 6-cutter may not yield a fragment
sufficiently small to amplify.
2) use very little DNA for the ligation.
3) use a nested primer for purification
a sample protocol may be:
a. design primer pairs so that several 6-base cutters do not cleave
between them. primers should direct replication away from each other.
b. cleave chromosomal DNA to completion with at least 6 6-base cutters
chosen as above. i cleave about 1 microgram E. coli DNA in 20 ul.
c. dilute and ligate to from circles; use a concentration of less than
0.005 micromolar ends to ensure monomer circles. unlike a typical
cloning ligation, you don't want dimers.
d. precipitate and dilute for the PCR
e. purify PCR products and reamplifiy use 1 or more nested primers;
this will eliminate any spurious bands.
f. sequence from both ends of the PCR product. the join point is
identifyable as the restriction site.
i've done it using a 4-base cutter and a partial digestion. its
disconcerting in that you always get a smear of products of mutiple
sizes. the sequence looks good until the first restriction site. it
then decays as you read both "proper" sequence and "improper" sequence
as you read across the join point in some fraction of your template.
ieally, a partial digestion with a 5-base cutter is ideal, but i never
tried it.
