Portal de Periódicos da CAPES

Human Duplex Sex Determination PCR

2001; Future Science Ltd; Volume: 31; Issue: 1 Linguagem: Inglês

10.2144/01311bm03

1940-9818

Bert Gold, Julie Bergeron, Marcia Triunfol, Michael Dean,

Molecular Biology Techniques and Applications

used in combination with any other M13-tailed forward primer.We then tested whether the method could be extended to analysis on an automatic capillary sequencer.Three M13 primers end-labeled with WellRED fluorescent dyes D2, D3, and D4 (Beckman Coulter, Fullerton, CA, USA) were ordered from Research Genetics (Huntsville, AL, USA).These primers were used for the amplification of labeled alleles using the same protocol described previously for 32 P-labeled primers.After amplification, the PCR products were pooled (i.e., up to nine different products, three size ranges, and three dyes).For desalting, wells of a MultiScreen  -FB filter plate (Millipore, Bedford, MA, USA) were pre-wetted with binding buffer [7 M guanidine-HCl and 200 mM 2-(N-morpholino) ethanesulfonic acid, pH 5.6], the pooled PCR products were then mixed in a 1:1 ratio with binding buffer and loaded onto individual wells by centrifugation at 1000× g for 5 min.The wells were then washed twice with 80% ethanol, and the products were eluted with 50 µL TE buffer (0.1 mM EDTA and 10 mM Tris-HCl, pH 8.0).Fifteen microliters of eluate were mixed with 25 µL deionized formamide and 0.5 µL size standards (Beckman Coulter) labeled with WellRED fluorescent dye D, then loaded and analyzed on a CEQ 2000XL capillary DNA analysis system (Beckman Coulter).Figure 2 shows an analysis of a run containing six multiplexed PCR products.Genomic DNA from strain A amplified by three markers along with M13 primer end-labeled with WellRED fluorescent dye D2 is compared with genomic DNA from strain B amplified with the same markers along with M13 primer end-labeled with fluorescent WellRED dye D3.As observed with 32 P-labeled markers, PCR products migrate as clean-laddered fluorescence peaks without interfering artifactual bands and, thus, allow clear identification of the origin of the alleles.We have obtained similar results when multiplexing was initiated at the level of PCR, by amplifying genomic DNA with several primer combinations simultaneously.It is reasonable to anticipate that this method could be used with dyes designed for other sequencing platforms.For instance, M13 primers labeled with