Genotyping Analysis Of Multiple Loci In Single Cell By Primer Extension Preamplification And Degenerate Oligonucleotide Primed-PCR

Preimplantation genetic Diagnosis (PGD) allows selective transfer of unaffected embryos using artificial reproductive techniques and single cell genetic analysis, enables genetic diagnosis to be offered before implantation, thus avoiding the need for termination of pregnancies in couples at risk for having children with genetic and chromosomal disorders. Because PGD are performed on single cell, genetic information is limited by either polymerase chain reaction (PCR) or fluorescent in-situ hybridization (FISH). Therefore, it is almost impossible to detect multiple genetic loci simultaneously or confirm the same locus repeatedly.Whole genomic amplification (WGA) can amplify all genomic sequence from minute DNA samples even from a single cell, provide sufficient DNA templates for a number of subsequent PCR amplification. Implication of WGA for PGD, the number of loci studied can be substantially increased and the genotyping can be confirmed.WGA has various strategies such as primer extension preamplification (PEP) and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). In PEP a mixture 15-base random oligonucleotides to prime Taq DNA synthesis frequently throughout the genome. DOP-PCR uses a primer 5 ' -CCGACTCGAGNNNNNNATGTGG-3' , two step PCR strategy consists of several low temperature annealing and extention to tagged many binding sites in the genome , and followed by more specific at the fragments tagged with the above primer sequence and this process is accomplished by increasing the annealing temperature, allows an unselected amplification of any source of DNA. A mixture random oligonucleotides and the uniform PCR temperature cycling perform in both PEP and DOP-PCR, in this condition, the efficiency of every primer annealing and extention is different. The coverage and yield of WGA may be different.It is reported that PEP from a single cell has implicated for ZFX/ZFY, Tay Sachs Disease , Cystic Fibrosis , Hemophilia A, Duchenne muscular dystrophy , RliD blood type and some short tandem repeat (STR). DOP-PCR from a single cell has been used mainly for cytogenetic studies by subsequent comparative genomic hybridization(CGH ) , is seldom for genetic studies by sequent PCR. Combining cytogenetics by CGH with genetics by PCR, DOP-PCR has the potential to diagnosis simultaneously genetic and chromosomal disorders.I Genotypic analysis of multiple loci in single cell by PEP protocols Objective To establish a technique of PEP from single cell followed by nest PCR for the detection of CD17 and nt-28 for P -thalassemia and ATTTT repeat linked with P -thalassemia, STR D18S51, D21S11 and D21S1411 genes. Thesimultaneous detection using PEP-nest PCR for the above loci with CFAF508 for cystic fibrosis and linked GATT repeat, DMD17 and 48 for Duchenne muscular dystrophy, SRY, sex-determination gene of chromosome Y for was carried out for examination of the coverage of the techniques. Methods Single lymphocyte in normal males or female and single blastomere from couple with no family history of genetic disorders were obtained. Using 15-base random primers, the entire genome of a single cell was amplified by primer extension preamplification (PEP). The 10 specific genetic loci were amplified from a small aliquot of the PEP reaction by nest PCR. Results: 1. PEP from single cell followed by nest PCR for the detection of CD17 and nt-28 for P -thalassemia and ATTTT repeat linked with P -thalassemia, STR D18S51, D21S11 and D21S1411 genes was successfully established. 2. The simultaneous detection using PEP-nest PCR for the above loci with CFAF508 for cystic fibrosis and linked GATT repeat, DMD17 and 48 for Duchenne muscular dystrophy, SRY, sex-determination gene of chromosome Y for was carried out, and the coverage of the genome from single lymphocytes is 97.17 %(583/600), single blastomere is 91.58% (174/190). ㏕he coverage of single blastomere is significantly lower than that of the single lymphocyte, P<0.05.II Genotypic analysis of multiple loci in single cell b...