In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level

Anal Biochem. 2008 Dec 15;383(2):151-8. doi: 10.1016/j.ab.2008.08.011. Epub 2008 Aug 20.

Abstract

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.

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / genetics
  • Cell Line
  • Chromosomes, Human / genetics
  • DNA / analysis*
  • DNA / chemistry*
  • DNA / genetics
  • DNA / isolation & purification
  • DNA Ligases / genetics
  • DNA-Binding Proteins / genetics
  • Gels*
  • Genomics
  • Genotype
  • Haplotypes
  • Humans
  • Molecular Weight
  • Nucleic Acid Amplification Techniques / methods*
  • Peptide Hydrolases / genetics
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide
  • Protein Serine-Threonine Kinases / genetics
  • Sepharose*
  • Temperature
  • Time Factors
  • Tumor Suppressor Proteins / genetics

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Gels
  • Tumor Suppressor Proteins
  • DNA
  • Sepharose
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein Serine-Threonine Kinases
  • Peptide Hydrolases
  • DNA Ligases