PCR techniques

Some of the 32 (known?) PCR techniques:
·       Allele specific PCR,
·       Alu PCR,
·       Asymetric PCR,
·       Colony PCR,
·       Competitive quantitative PCR.
·       Competitive RT-PCR (c-RT-PCR).
·       Differential display RT-PCR.
·       Hot start PCR,
·       Immuno-PCR on chip
·       In Situ PCR.
·       Inverse PCR,
·       ISSR PCR,
·       Late PCR,
·       Ligation anchored PCR (LA-PCR).
·       Long PCR,
·       Long PCR,
·       Marathon RNA PCR.
·       Multiplex PCR,
·       Multiplex PCR.
·       Nested PCR,
·       Non-competitive RT-PCR.
·       PCR cloning,
·       PC sequencing
·       RACE-PCR.
·       RAP-PCR (RNA arbitrarily primed PCR).
·       Real time PCR
·       RNase ligase mediated PCR (RLM_RACE PCR).
·       RT PCR,
·       Standard PCR,
·       Suppression subtraction Hybridization PCR (SSH).
·       Touch down PCR,
·       AFLP PCR,
·       Single cell PCR

454 pyrosequencing

As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the pyrogram (intensity).

actual sequence: GCAGGCCT


http://genoseq.ucla.edu/action/view/Pyrosequencing

Amplification Refractory Mutation System (ARMS)

also called allele-specific PCR (ASP)
to amplify or detect mutant DNA, use 3 primers

ARMS has the ability to isolate low levels of a mutant sequence in a background of wild-type DNA. The system depends on the specificity of a primer for the normal sequence and another primer for the mutation.

In ARMS, the primer pair is designed so that one of the 3' ends coincides with a variant nucleotide in the target sequence. When the primer mismatches the template the frequency of extension is very low and consequently the effective number of sequence copies available for amplification is greatly reduced.

The principle of ARMS. Primers are designed to amplify wild-type and mutant sequence. The difference between these two primers is the 3' base, where one matches wild-type sequence and the other the mutant. The reaction containing the mutant primer will only extend mutant template and wild-type template will not be extended by this primer. (3' base of primer is where extension begins, therefore need specificity)

http://www.horizonpress.com/pcr/pdf/rtpcr/rtpcr09.pdf
http://clincancerres.aacrjournals.org/content/12/13/3875/F1.large.jpg

high resolution melting analysis

high resolution melting analysis (HRM):
-used to detect SNP
-must use a saturating dye (e.g. Evagreen)
-after real-time PCR, normalize a melt curve, suppress the curve that differs the most.

Methylation Sensitive (MS)-HRM:

-Bisulfite treatment of DNA converts unmethylated Cytosine to Uracil.
— Methylated Cytosine are protected and remain intact.

-After PCR amplification all Uracils are converted Thymine.

-perform DNA sequence analysis, if T, that means the DNA is unmethylated.
if C, the DNA is methylated.