PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.
AUTOR(ES)
Warburton, P E
RESUMO
Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymerase chain reaction (repPCR), we are able to amplify a representative sampling of multiple repetitive units simultaneously, allowing rapid analysis of chromosomal subsets. Direct sequence analysis of repPCR amplified alpha satellite from chromosomes 17 and X reveals positions of sequence heterogeneity as two bands at a single nucleotide position on a sequencing ladder. The use of TdT in the sequencing reactions greatly reduces the background associated with polymerase pauses and stops, allowing visualization of heterogeneous bases found in as little as 10% of the repeat units. Confirmation of these heterogeneous positions was obtained by comparison to the sequence of multiple individual cloned copies obtained both by PCR and non-PCR based methods. PCR amplification of alpha satellite can also reveal multiple repeat units which differ in size. Analysis of repPCR products from chromosome 17 and X allows rapid determination of the molecular basis of these repeat unit length variants, which appear to be a result of unequal crossing-over. The application of repPCR to the study of tandemly repeated DNA should allow in-depth analysis of intra- and interchromosomal variation and unequal crossing-over, thus providing insight into the biology and genetics of these large families of DNA.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=334470Documentos Relacionados
- Molecular analysis of a deletion polymorphism in alpha satellite of human chromosome 17: evidence for homologous unequal crossing-over and subsequent fixation.
- Interchromosomal Effects and the Relation between Crossing-over and Nondisjunction
- Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.
- Suppression of crossing-over by DNA methylation in Ascobolus
- Interstitial duplication/deletion owing to unequal crossing-over in association with pericentric inversion.