| Objectives To evaluate nested PCR and PEP-nested PCR in a single cell to determine the ability to accurately amplify and correctly diagnose a targeted gene, and to establish the condition of the single-cell PCR , and to apply PEP-nested PCR to sexing in a single pre-embryo blastomere.Methods 1) To test whether the SRY and ZP3 gene primers selected were the correct choice for the test and the sensitivity and specifity of conventional PCR and nested PCR. There were five healthy males, whose karyotype were 46,XY, and five healthy females who never fall pregnant, whose karyotype were 46,XX. They were took out 3ml whole blood from median cubital vein. DNA was extracted from 1 ml whole blood of theirs and continuously diluted the solution of DNA to density to be 6pg/μL. a: 400ng of individual human blood DNA template in a final volume of 50μl was carried out conventional PCR with SRY gene and ZP3 gene inner or outer primers. b: 100 ng, 10 ng, 1 ng, 100 pg of individual human blood DNA template were carried out conventional PCR with SRY gene and ZP3 gene inner primers. c: 10 ng, 1 ng, 100 pg, 10 pg, 6 pg of individual human blood DNA template were carried out nested PCR with SRY gene and ZP3 gene primers. 2) To test the precision, sensitivity, and stability of PCR amplification with the designed primers and to compare the allele dropout (ADO) rate of SRY, amplification rate and correct diagnosis rate of nested PCR and PEP-nested PCR. Additionally 2 ml of whole blood were centrifuged and made into suspension of lymphocyte with PBS. The single lymphocyte was individually aspirated into a fine han-drawn Pasteur pipette under an inverted microscope.Sixty-four male and sixteen female single lymphocyte were collected and divided randomly into two groups(n=40/group): a: amplified SRY and ZP3 gene by nested PCR . b: amplified whole genome by 15-base random primers(PEP), then a small aliquots of PEP product(5μl) were re-amplified SRY and ZP3 gene by nested PCR. Positive control group ( 5 lymphocytes group ) and negative control group ( blank group ) were established and amplificed at the same time while single cell was biopsied. The ADO rate of SRY, amplification rate and correct diagnosis rate of two amplifications were analyzed with exect method. 3) Two embryo were fertilized through IVF-ET and thawed. They were digested zona pellucida with Tryode's acid solution( PH 2. 2). Seven Single blastomere were collected and PEP-PCR in order to diagnose their sexuality.Results 1) After SRY and ZP3 gene were amplificed with conventional PCR, 100ng, 10ng, minimum to be 1 ng of the DNA template were positive for the testis-determining-gene and ZP3 gene(male) or ZP3 gene(female). And discovered that the quantity of amplification production correspondingly decreased along with the reduction of DNA template. But experimental conditions is easy to influence conventional PCR. Major problem is the quantity of DNA template. 2) When the quantity of DNA template was the order of 6 pg, which was approximately the amount present in a single diploid human cell, nested PCR can accurately amplificed SRY and ZP3 gene. It's sensitivity and specifity were 100%. So it can offered sensitive and specific detection methology for preimplantation genetic diagnosis. 3) After biopsied single lymphocyte amplificed with nested PCR and PEP-nested PCR, the allele dropout (ADO) rate of SRY gene by nested PCR and PEP-nested PCR were 15.63%, 3.13%, while amplification rate were 93.06%, 98.61%; correct diagnosis rate were 87.50%, 97.50% ,respectively. Though without statistical meaning(P=0.086), the ADO rate in PEP-nested PCR dropped nearly 5 timesthan that in nested PCR. The amplification rate and correct diagnosis rate also are obviously high in PEP-nested PCR. 4) After seven single pre-embryo blastomeres amplificed SRY and ZP3 gene with PEP-nested PCR, five blastomere from the pre-embryo were diagnosed male , whereas two from the other were female.Conclusions: 1) When SRY gene is targeted gene and ZP3 gene is co...
|
PDF Full Text Request