Producing STR Locus Patterns from Bloodstains and Other Forensic Samples Using an Infrared Fluorescent Automated DNA Sequencer

Abstract

Short tandem repeat (STR) analysis is increasingly being used in forensic case analysis because of the large number of STR loci in the human genome and their highly polymorphic nature. An automated DNA sequencer using high sensitivity infrared (IR) fluorescence technology was used to detect STR allele patterns from simulated forensic samples. The amplification strategy used a 19 base pair extension on the 5′ end of one of the PCR primers. This sequence is identical to the sequence of a universal M13 Forward sequencing primer which is included in the amplification reaction. Allelic bands were detected by incorporation of the M13 primer-fluorescent dye conjugate into PCR products thus eliminating the need for direct conjugation of fluorescent dye to individual STR primers. By using an IR-based automated DNA sequencer and Tth DNA polymerase, polymorphic STR alleles were detected on-line rapidly and efficiently from bloodstains using only a high temperature incubation to extract DNA from blood cells. Five STR loci were also amplified using Chelex extracted DNA from simulated forensic samples. Multiplexing of three primer pairs in a single PCR mixture for amplification was accomplished using Taq polymerase. This system combines IR fluorescence chemistry and laser technology thus eliminating the need for radioactivity and the gel handling required with silver staining and fluor detection systems. Real-time detection permits immediate visualization of the data and STR alleles are displayed as familiar autoradiogramlike images that can be analyzed by computer. By loading a 64 lane gel twice and multiplexing with three primer pairs, forensic scientists can type at least three loci from 120 samples in one day.

Quantitative trait loci (QTLs) analysis of palm oil fatty acid composition in an interspecific pseudo-backcross from Elaeis oleifera (H.B.K.) Cortés and oil palm (Elaeis guineensis Jacq.)

“…Our choice favored the SSRs with best amplification patterns, a priori segregating from the SA65T and PO3228D parents, and with high polymorphism. The plant material was analyzed by the SSR technique in fluorescent conditions according to Roy et al (1996) using the capillary electrophoresis and detection systems of a 3500xL Genetic Analyzer (Applied Biosystems, USA). The PCR amplification was performed in a 384-well Eppendorf Mastercycler with 25 ng of DNA in a 10-μl final volume of buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.001 %w/v glycerol,) containing 1.50 mM MgCl 2 , 0.08 μM of the forward M13-tailed primer, 0.1 μM of the reverse primer, 200 μM of dNTP, 1 U of Taq DNA polymerase (Life Technologies, USA), and 0.1 μM of M13 primer-fluorescent dye FAM, NED, PET, or VIC (Applied Biosystems, USA).…”

Development of microsatellite markers in Cannabis sativa for DNA typing and genetic relatedness analyses

“…After examining six different temperatures, the optimal annealing temperature was determined to be 53°C at which the amplicons had the highest intensity as measured by relative fluorescence units (rfu). With some modifications, the M13 fluorescent tail primer method described by Roy et al (1996) [27] was used as a screening technique to detect polymorphism among 25 different loci. Fragment analysis was performed using either the method described by Roy [27] or direct 5'-labeled fluorescent primers.…”

Sequence Analysis of Mitochondrial DNA Hypervariable Regions Using Infrared Fluorescence Detection

“…IR fluorescent visualization of amplification products is facilitated by covalently attaching a universal M13 primer sequence "tail" on the 5 ′ end of one or both amplification primers, as described previously (11). M13 tails utilized in this work were M13 Forward (-29) (5 ′ -CACGACGTTGTAAA-ACGAC-3 ′ ) (14) and M13 Reverse (5 ′ -GGATAACAATTTC -ACACAGG-3 ′ ).…”

“…We have previously used polymerase chain reaction (PCR) amplification coupled with infrared (IR) fluorescence detection (7) for analysis of short tandem repeat (STR) polymorphisms (11,13), determination of gender (10) and typing of D1S80 alleles (9). Several potential strategies exist for amplification and sequence analysis of human HV1 and HV2.…”