In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.
The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.
Michikawa
Yuichi
Y
RadGenomics Project, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Inage, Chiba 263-8555, Japan.
Sugahara
Keisuke
K
Suga
Tomo
T
Ohtsuka
Yoshimi
Y
Ishikawa
Kenichi
K
Ishikawa
Atsuko
A
Shiomi
Naoko
N
Shiomi
Tadahiro
T
Iwakawa
Mayumi
M
Imai
Takashi
T