Multiplex amplification of short tandem repeat loci
A short tandem repeat, multiple amplification technology, applied in recombinant DNA technology, microbial assay/inspection, instrument, etc., can solve the problem of undeveloped materials or methods for multiple amplification of 13 or more STR loci, etc. , to save time
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Embodiment 1
[0131] Fluorescence detection of locus D3S1358 with ABI PRISM(R) 310 Genetic Analyzer, HUMTH01, D21S11, D18S51, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, and HUMCSF1PO multiple amplification of
[0132] In this example, each locus D3S1358, HUMTH01, D21S11, D18S51, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, and HUMCSF1PO of a DNA template was simultaneously amplified in one reaction tube. PCR amplification was performed in 25 μl 1×Gold ST*R buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25°C), 0.1% Triton X-100, 1.5 mM MgCl 2 , 160μg / ml BSA and 200μM each of dATP, dCTP, dGTP, and dTTP), with 1ng template and 3.25U AmpliTaq Gold TM DNA polymerase is carried out. The following amplification protocol was used in a GeneAmp PCR System 9600 (Perkin Elmer, Poster City, CA): 96°C for 12 minutes; then 10 cycles of 94°C for 30 seconds, ramp to 58°C in 68 seconds, hold for 30 seconds, Gradually ramp to 70°C within 50 seco...
Embodiment 2
[0138] Fluorescence detection of locus D3S1358 with ABI PRISM(R) 310 Genetic Analyzer, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, Multiplex amplification of D16S539, HUMCSF1PO and S159
[0139] In this example, each locus D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO of a DNA template was simultaneously amplified in one reaction tube and S159. PCR amplification was performed in 25 μl 1×Gold ST*R buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25°C), 0.1% Triton X-100, 1.5 mM MgCl 2 , 160 μg / ml BSA and 200 μM each of dATP, dCTP, dGTP, and dTTP) with 1 ng template and 4 U AmpliTaq Gold TM DNA polymerase is carried out. The following amplification protocol was used in a GeneAmp PCR System 9600 (Perkin Elmer, Poster City, CA): 96°C for 12 minutes; then 10 cycles of 94°C for 30 seconds, ramp to 58°C in 68 seconds, hold for 30...
Embodiment 3
[0145] Fluorescence detection of locus D3S1358 with ABI PRISM(R) 377 DNA sequencer, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, Multiplex amplification of D16S539, HUMCSF1PO and S159
[0146] In this example, DNA samples were amplified as in Example 2. Amplified products were separated using an ABIPRISM(R) 377 DNA sequencer. This is with 0.2mm thick 5% Long Ranger TM Acrylamide (FMC BioProducts, Rockland, ME), 7M urea gels were performed. DNA samples were mixed with 1.5 μl of loading buffer (88.25% formamide, 4.1 mM EDTA, 15 mg / ml blue dextran) and 0.5 μl of internal lane size standard, denatured at 90°C for 2 min, and removed before loading Pre-chill on ice. Electrophoresis was performed using the manufacturer's GeneScan(R) pre-electrophoresis module (PRGS 36A-2400) and electrophoresis module (GS 36A-2400). The electrophoresis time is 3 hours and the effective filter A is used.
[0147] Figure 3A is a...
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