Quantitative effects of position and type of single mismatch on single base primer extension
. J Microbiol Methods.
For DNA amplification, the C1000TM Thermo Cycler Bio-Rad, Germany, was programmed under the conditions involving denaturation at 94[degrees]C for 5 min; 30 cycles of denaturation at 94[degrees]C for 1 min, primer annealing at 52[degrees]C for 45 Sec and primer extension
at 72[degrees]C for 2.5 min; final extension step at 72[degrees]C for 10 min.
This can be solved by real-time analysis, but there is no guarantee that the assay is optimized or that the PCR results are attributable to primer extension
Testing was completed on the Tm Bioscience/Luminex Universal Array platform using primer extension
Thermo cycling consisted of an initial denaturation at 94AdegC for 2 min followed by 35 cycles of denaturing at 94AdegC for 1 min, primer annealing (58-65AdegC) for 2 min and primer extension
(72AdegC) for 2 min.
PCR mixture preparation and conditions: The reaction mixture (25 ul) was prepared by adding 2 ul forward primer, 2 ul reverse primer, 6 ul DNA, 12.5 ul master mix solution and 2.2 ul water free nuclease for full length 16S rRNA gene amplification and initially was denatured at 94degC for 2 min followed by 30 cycles consisting of denaturation at 94degC for 60 s, primer annealing at 55degC for 60s and primer extension
at 72degC for 3 min and a final extension at 72degC for 10 min using multigene thermal cycler.
Eppendorf Master Cycler was utilized to carry out PCR reactions with initial denaturation at 94[degrees]C for 3 minutes, trailed by repeated cycles of denaturation at 94[degrees]C for 45 seconds, annealing as per the primer's melting temperature for 1 min and primer extension
at 72[degrees]C for 1 min.
The conditions used in one step RT-PCR were include reverse transcription with the Incubation at 45degC for 45 min, initial denaturation at 95degC for 5 min, and 40 cycle each of 95degC for 40 sec, annealing at 56degC for 1 min, primer extension
at 72degC for 30 sec followed by final extension at 72degC for 10 min.
Reactions of the primer extension
were performed using the SNaPshotddNTP Primer Extension
Kit (Applied Biosystems, Foster City, CA, USA).
The SNaPshot primer extension
products are separated with
Then, for the samples that cannot be haplotyped with the allele-specific primer, the sequencing primers are selectively blocked with dideoxyribonucleoside triphosphate (ddNTP) to restrict the designated primer extension
, and the haplotypes of the PCR products are identified by sequencing.
First, primer extension
is performed using site-specific primers and a combination of dNTPs and ddNTPs, selected so that different alleles will result in products of different and predicted sizes.