Alu-based vectorettes and splinkerettes. More efficient and comprehensive polymerase chain reaction amplification of human DNA from complex sources.
Alu-polymerase chain reaction (PCR) is widely used to amplify human specific fragments from complex heterologous DNAs, such as somatic cell hybrids or yeast artificial chromosome (YAC) recombinants, but the fragments amplified are limited in number and are nonrepresentative. This report describes a modified one-sided alu-PCR technique, which offers better representation of amplified sequences while maintaining human specificity. The method relies on the ligation of partially mismatched double-stranded oligonucleotides (vectorettes or splinkerettes) to endonuclease-restricted DNA and universal priming with a single alu-consensus primer, the complement to which is the unpaired region. Alu-vectorette and alu-splinkerette-PCR of two somatic cell hybrids results in a greater complexity of products than alu-PCR alone. The advantage of alu-splinkerette over alu-vectorette-PCR is the elimination of nonspecific priming owing to the presence of the vectorette primer and to an increase in the product size range, a consequence of the difference in the splinkerette design. Alu-splinkerette-PCR is a useful technique for generating new and more comprehensive markers of the human sequences contained in somatic cell hybrids and YACs.
Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, United Kingdom.
S J
SJ
Qureshi
D J
DJ
Porteous
A J
AJ
Brookes
D001483:Base Sequence; D018244:Chromosomes, Artificial, Yeast; D004247:DNA / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid
D004247:DNA