Protocol Online logo
Top : Forum Archives: : Real-Time PCR

allele-specific amplification - (Aug/23/2006 )


what is meant by allele-specific amplification ( in lab work terms)???

is it the same as ASO ( allele-specific oligonucleotide ) ??


allele-specific PCR - is the same as ARMS or MAMA-PCR and has a lot of
variations and kits - for example Amplifluor Primer ( previously SunRise univ primer)(endpoint)
GC-tailed- primers with melting curve(endpoint)- Blunt-ended hairpin primers with sybr(realtime)
Tetraprimer ARMS (gel)etc

smile.gif detection SNPs or mutations by 3' mismatch differential extention by Taq (Stoffel Klentaq)
in oder to improve discrimination 3' can be additionally destabilized
but in early days mismatch was also used in the midle of primer for as-pcr

with slow cyclers and long holds mismatch is also amplified sad.gif
Additionally Multiplex as-pcr with labelled primers + gel
As a result HotStart+Touchdown(stepdown)+hairpin+ fast cycling = MAXIMAL DISCRIMINATION

1: Clin Chem. 1993 May;39(5):804-9. Links
Rapid cycle allele-specific amplification: studies with the cystic fibrosis delta F508 locus.
Wittwer CT, Marshall BC, Reed GH, Cherry JL.

Rapid cycle DNA amplification is a polymerase chain reaction technique with improved product specificity and cycle times of 20-60 s, allowing complete 30-cycle reactions in 10-30 min. The presence or absence of the delta F508 deletion and wild-type allele was determined in 104 cystic fibrosis patients by rapid cycle DNA amplification. In separate allele-specific assays, sequences on both sides of the delta F508 locus were amplified with the 3' end of a discriminating primer at the delta F508 locus, with either a 3-bp or a 1-bp mismatch. With rapid cycling (35-s cycles), single-base discrimination was achieved over a broad range of annealing temperatures (50 degrees C or lower); with conventional cycling and "hot starts" (160-s cycles), only annealing temperatures of 61-62 degrees C sufficiently discriminated between alleles. With rapid cycling, genotype could still be assessed with annealing temperatures as low as 25 degrees C. We conclude that faster temperature cycling can improve the results of allele-specific amplification.