An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening.
We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 degrees C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in three different techniques of genomic analysis: comparative genomic hybridization (CGH), genotyping at simple tandem repeat (STR) loci and screening for single base mutations in a common monogenic disorder, beta-thalassemia. gDNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues can also be amplified with this protocol.
Lee
Cheryl I P
CI
Division of Medical Sciences, National Cancer Centre, 11 Hospital Drive, Singapore 169610.
Leong
Siew Hong
SH
Png
Adrian E H
AE
Choo
Keng Wah
KW
Syn
Christopher
C
Lim
Dennis T H
DT
Law
Hai Yang
HY
Kon
Oi Lian
OL
D022202:Chromosomes, Artificial, Bacterial; D004247:DNA; D017931:DNA Primers; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D017354:Point Mutation; D020080:Tandem Repeat Sequences; D013696:Temperature; D017086:beta-Thalassemia / Q000235:genetics
D017931:DNA Primers; D004247:DNA