Logo of narLink to Publisher's site
Nucleic Acids Res. Sep 11, 1990; 18(17): 5153–5156.
PMCID: PMC332136

Disruption of phase during PCR amplification and cloning of heterozygous target sequences.


PCR amplification of genomic DNA or cDNA has become a standard tool for identification of mutations underlying genetic disease. There are inherent limitations in the application of this method in compound heterozygotes. One problem which is encountered is the disruption of phase (linkage) between heterozygous polymorphisms represented on heterologous alleles. A test system was used to demonstrate and quantitate the disruption of phase between two polymorphic restriction sites. Phase is disrupted in approximately 1% of the PCR amplified material, possibly due to incomplete chain elongations and subsequent priming on the heterologous allele. Phase is disrupted in approximately 1/4 of cloned PCR fragments, possibly due to excision repair of heteroduplexes during cloning. The implications of these disruptions for the use of PCR in identifying mutations are discussed.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (830K), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ledley FD, Jansen R, Nham SU, Fenton WA, Rosenberg LE. Mutation eliminating mitochondrial leader sequence of methylmalonyl-CoA mutase causes muto methylmalonic acidemia. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3147–3150. [PMC free article] [PubMed]
  • Jansen R, Kalousek F, Fenton WA, Rosenberg LE, Ledley FD. Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction. Genomics. 1989 Feb;4(2):198–205. [PubMed]
  • Ledley FD, Crane AM, Lumetta M. Heterogeneous alleles and expression of methylmalonyl CoA mutase in mut methylmalonic acidemia. Am J Hum Genet. 1990 Mar;46(3):539–547. [PMC free article] [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Scharf SJ, Horn GT, Erlich HA. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. [PubMed]
  • Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. [PMC free article] [PubMed]
  • Päbo S, Irwin DM, Wilson AC. DNA damage promotes jumping between templates during enzymatic amplification. J Biol Chem. 1990 Mar 15;265(8):4718–4721. [PubMed]
  • Pukkila PJ, Peterson J, Herman G, Modrich P, Meselson M. Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli. Genetics. 1983 Aug;104(4):571–582. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...