DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies.

DNA testing from mixed cell samples can be difficult to use successfully in criminal investigations. Here, we present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody-microbead captured cell separation. This method, together with the multiplex short tandem repeat typing presented, has proven highly successful in the recovery of DNA profiles corresponding to the ABO blood type. Methodological steps include magnetic separation using leukocyte specific CD45 antibody-coated microbeads and centrifugal separation of leukocyte agglutination by ABO antibody. The detection results of variable mixed ratio showed that the target DNA was detected accurately as low as 1:512 mixed ratio, regardless of the large amount of the background DNA present. The method presented here is applicable to PCR-based identification for various kinds of mixed samples.

Vanadium accelerates polymerase chain reaction and expands the applicability of forensic DNA testing.

Polymerase chain reaction (PCR) has been rapidly established as one of the most widely used techniques in molecular biology. Because most DNA analysis is PCR-based, the analysis of unamplifiable DNA of poor quality or low quantity is nearly impossible. However, we observed that if an appropriate concentration of vanadium chloride is added to the standard reaction mixture, the enzymatic amplification of DNA could be enhanced. Using multiplex PCR with the addition of vanadium, DNA typing was possible from even trace amounts of DNA that we were unable to amplify using normal reaction conditions. This method might be an effective tool for not only criminal investigations and ancient DNA analysis, but also for nearly all fields using DNA technology.

Application of a novel multiplex polymerase chain reaction system for 12 X-chromosomal short tandem repeats to a Japanese population study.

The analysis of X-chromosomal short tandem repeat (X-STR) polymorphisms has been the focus of attention in several researches, mainly due to its applicability in the investigation of complex kinship cases. A new 12 X-STR multiplex system (GATA172D05, DXS7423, DSX6809, DXS10134, DXS7132, DXS9902, DXS6789, DXS10074, DXS8378, DXS9898, DXS10147, and GATA31E08) was developed and applied to a Japanese population study. DNA samples from 290 males and 160 females were successfully analyzed using the 12 X-STR multiplex system. No mutation was detected in the kinship cases involving 34 family trios. The combined powers of discrimination of the 12 X-STR loci in males and females were 0.999997 and 0.9999999996, respectively. We conclude that the combined analysis of 12 X-STR loci using this single multiplex polymerase chain reaction system is a powerful tool in forensic DNA testing.