Reena Roy, PhD1, David L. Steffens, PhD2, Bill O. Gartside2,
Gi Y. Jang, PhD2 and John A. Brumbaugh, PhD2
1Nebraska State Patrol, Lincoln, NE 68502
2LI-COR, Inc., Lincoln, NE 69504
Restriction fragment length polymorphism (RFLP) analysis is the currently accepted method for individualizing blood and body fluid stains from various samples in forensic laboratories. Since samples may be degraded or limited in quantity, they may not always be suitable for analysis by this conventional method. Amplification of such samples by the Polymerase Chain Reaction (PCR) method may yield information as to the genetic identity of individuals. The discovery of highly polymorphic short tandem repeats (STRs) has proven very useful for medical diagnostics, genomic mapping and forensic analysis.
The purpose of this research was to use an automated detection system using laser irradiation along with PCR primers labeled with an infrared (IR) fluorophore for detection of PCR amplified STRs from minute quantities of dried bloodstains. STR loci chosen for analysis were ACTBP2, HUMTHO1, D2S436, D18S535 and D20S470. A mixture consisting of Tth enzyme buffer plus the forward and reverse primers was prepared for each bloodstain. This mixture was aliquoted into tubes containing a single bloodstained thread approximately 1 mm in length. The forward primer pair of each contained a 5' end tail identical to a universal M13 forward primer sequence facilitating fluorescent labeling of the PCR products during amplification. The tubes were incubated at 92ºC for 20 minutes in a Perkin-Elmer GeneAmp PCR System 9600. A second mixture consisting of Tth DNA polymerase, dNTPs and IR-labeled M13 forward primer was prepared and added to the tubes at the end of the high temperature incubation while tubes were still at 92ºC. The reactions were cycled at 94ºC for 20 seconds and 62ºC for 1 minute for 27 cycles. Stop solution was added to each tube and samples were diluted and loaded onto the gels. Seven percent denaturing Long-Ranger gels run at 2000V (constant) were used to resolve the STR bands. A separation distance of 15 cm produced rapid migration of the DNA fragments and run times of less than one hour from sample loading to detection of DNA fragments up to 350 bases in length.
To validate the results obtained with unextracted bloodstains, DNA was extracted from bloodstains, saliva and hair samples and subjected to amplification. STR alleles were detected and displayed the same binding patterns from purified DNA as from bloodstains using either Tth or Taq polymerase. For multiplexing two additional unlabeled amplification primers (one containing the M13 tail) were added to the reaction mixture for each additional locus to be analyzed. Multiplexing of three primer pairs in a single PCR amplification mixture was accomplished using Taq polymerase.
By using the infrared fluorescence automated DNA sequencer and Tth DNA polymerase, the polymorphic STR alleles were detected rapidly and efficiently from bloodstains without prior DNA extraction. This system combines infrared fluorescence chemistry and laser technology and thus eliminates the necessity of using radioactivity. Allelic bands are detected by incorporation of the M13 primer-fluorescent dye conjugate into the PCR products which eliminates the need for direct conjugation of fluorescent dye to STR primers. The STR alleles are displayed as familiar autoradiogram-like bands. By using the described method and multiplexing strategies it is possible to generate STR images of at least 3 loci for up to 120 individuals in less than a day.
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