Sequencing using capillary electrophoresis of short tandem repeat alleles separated and purified by high performance liquid chromatography.
Polymerase chain reaction (PCR) amplified alleles need to be isolated and purified before carrying out additional analysis to confirm sequence, number of repeats and microvariants within a short tandem repeat (STR) locus. Also, PCR amplification of tetranucleotide repeat loci, used in DNA typing assays, often result in heteroduplex formation, adding to the complexity of analysis. Sequencing reactions require single specific target DNA for reliable sequencing analysis. Alkylated poly(styrene-divinylbenzene) columns at elevated temperature and gradient elution conditions increase the efficiency of separation to allow for the purification of PCR products. Using the separation technique of ion-pairing reverse-phase (IPRP) high performance liquid chromatography (HPLC), molecular biologists can separate and purify DNA fragments without alteration to the double-stranded DNA sequencing properties. In this study, the IP-RP chromatography technique has been demonstrated by separation of alleles of the short tandem repeat loci of TH01, vWA31, F13A01 and FES/ FPS. Alleles differing in size range of 12 to 4 base pairs were separated by IPRP/HPLC and individual alleles were peak-captured, then cycle-sequenced. These HPLC fractions required no additional steps prior to cycle sequencing. Capillary electrophoresis (CE) was used to sequence the alleles. Furthermore, CE offers advantages over traditional slab methods via automation and higher applied voltages. Interestingly, unlike traditional gel electrophoresis, samples were introduced into the sieving matrix by electrokinetic injection, which allows for multiple injections from a single sample, a key feature for method development. Applied voltage was 320 V per centimeter using a nonderivatized fused silica capillary with an interior diameter of 50 microm and a total length of 47 centimeters. The total analysis time including capillary filling and pre-electrophoresis was less than 30 min for a 220-bp fragment. A sequencing rate of 530 bp/h was achieved using these conditions. By combining the techniques of HPLC separation and CE sequencing, the results confirmed the sequence and number of nucleotide repeats for each STR loci. An average sequencing efficiency of 97% was achieved. Additionally, this method defined the absence of a 9.3 microvariant for a TH01 heterozygous individual previously typed as a 9, 9.3/10 using slab gel electrophoresis. The techniques described can be applied to other DNA purification and isolation problems.
Marino
M A
MA
Center for Medical and Molecular Genetics, Armed Forces Institute of Pathology, Washington, DC, USA. marino@email.afip.osd.mil
Devaney
J M
JM
Smith
J K
JK
Girard
J E
JE
D000483:Alleles; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid
D004247:DNA