Preliminary exploration of the influence factors on amplification of single cell duplex-nested PCR
- VernacularTitle:单细胞二重巢式PCR扩增影响因素初探
- Author:
Changgao ZHONG
;
Luyun LI
;
Changfu LU
;
Ge LIN
;
Guangxiu LU
- Publication Type:Journal Article
- Keywords:
Polymerase chain reaction;
Preimplantation diagnosis;
Beta-thalassemia
- From:
Journal of Peking University(Health Sciences)
2004;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the influence factors on amplification of single cell duplex-nested PCR.Methods:The mutational loci region CD41-42 and IVS-Ⅱ654 of ?-globin gene were amplified by duplex-nested PCR with different combination of primers concentration, different Taq DNA polymerases, different neutralization buffers and with or without predenaturation at 98 ℃ before the PCR amplification in single lymphocyte or single blastomere, thus, to investigate the influence of these factors on the amplification efficiency of PCR.Results:TaKaRa EX Taq was the most efficient Taq DNA ploymerase among different Taq DNA ploymerases; primer pair R1+F1 at final concertration of 0.25 ?mol/L and R2+F2 at 0.3 ?mol/L were the most efficient ones in amplification among different combinations of primers concentrations; the amplification efficiency in neutralization buffer-1 (200 mmol/L Tricine) was obviously higher than that of neutralization buffer-2 (900 mmol/L Tris-HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)(P0.05). Conclusion:There were remarkable differences of the amplification efficiency of single cell duplex-nested PCR while using different combination of primers concentrations, different Taq DNA polymerases, different neutralization buffers. However, predenaturation at 98 ℃ before the single cell PCR amplification could not improve the PCR amplification efficiency
