Seung Bum Seo

Technical Note: Efficiency of Total Demineralization and Ion-Exchange Column for DNA Extraction from Bone

We investigated whether a combination of recently introduced methods, total demineralization and ion-exchange columns, would increase DNA recovery from old bone. Ten bone samples taken after a burial period of approximately 60 years were used in this study. Bone powder was digested using total or incomplete demineralization. DNA was extracted by the standard organic method. The DNA extract was purified with ion-exchange columns or QIAquick spin columns. The efficiency of different DNA extraction methods was compared in terms of DNA concentration, inhibitors generated by real-time PCR, and conventional STR typing results. The mean DNA concentration using the total demineralization method is approximately 3 times higher than that using the incomplete demineralization method. For DNA purification, the method using QIAquick spin columns appeared to yield approximately double the DNA than the method using ion-exchange columns. Furthermore, 2 out of 10 samples showed higher levels of inhibition with C(T) values of IPC > or =30 cycles when using only ion-exchange columns. In STR results, total demineralization yielded more locus profiles by 4.2 loci than incomplete demineralization, and QIAquick spin columns also yielded more locus profiles by 3.5 loci than ion-exchange columns. Total demineralization of bone powder significantly increased DNA yield and improved STR typing results. However, the use of ion-exchange columns was not efficient when compared with the method using QIAquick spin columns. It is suggested that the combination of total demineralization and QIAquick spin columns lead to greatly improved STR typing results.

Autosomal short tandem repeat analysis of ancient DNA by coupled use of mini- and conventional STR kits.

Abstract:  Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpFℓSTR® MiniFiler™ kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpFℓSTR® MiniFiler™ and Identifiler™ kits. Whereas only 21.87% of larger‐sized loci profiles could be obtained with the Identifiler™ kit, 75% of the same loci profiles were determined by MiniFiler™ kit analysis. This very successful amplification of large‐sized STR markers from highly degraded aDNA suggests that the MiniFiler™ kit could be a useful complement to conventional STR kit analysis of ancient samples.

Improvement of short tandem repeat analysis of samples highly contaminated by humic acid

We investigated several methods for obtaining successful short tandem repeat (STR) results from high-humic acid (HA)-content samples. DNA purification efficiency was tested for QIAquick(®) PCR Purification, QIAamp(®) DNA Investigator and Prepfiler™ Forensic DNA Extraction kits. HA-removal capacity of Inhibitor Remover and InhibitEX(®) Tablet was tested. Experiments on overcoming HA effects on STR amplification were conducted using an AmpliTaq Gold(®) DNA Polymerase and a TaKaRa Ex Taq™ Hot Start Version (Ex Taq HS) with BSA addition. QIAquick kit was most efficient in HA removal and Ex Taq HS showed high resistance to HA. Increasing the amounts of Taq polymerases and BSA addition were shown to be efficient in overcoming PCR inhibition, but BSA addition was superior to the former method. Inhibitor Remover and InhibitEX(®) Tablet did not positively affect the STR results. This study will help achieve better STR results with high-HA-content samples.

Modification of a Commercially Available Kit for the Improvement of PCR Efficiency

The polymorphism of short tandem repeats (STRs) is commonly used for human identity testing. Many commercial kits are available for this purpose, in which multiplex PCR for 10-16 STRs is usually used. For optimal results, the kits recommend rather limited conditions, which not all forensic samples can satisfy. We increased the efficiency of several commercial kits by modifying the components and reaction parameters (primer concentration and cycle number, and annealing and extension time). To simulate low copy number, we used 1:10 and 1:100 diluted samples compared to the recommended concentration. A change in cycle number, annealing and extension time, and primer concentration showed various results. Fine-tuning of PCR conditions by combining the described changes, decreasing the primer concentration, and increasing the annealing and extension time together with increasing the cycling number dramatically increased the efficiency of the reaction, even in low-copy-number simulated samples. Detailed information for several kit components or kits with different components targeting difficult sample conditions, including low copy number, would be of great help in forensics.The polymorphism of short tandem repeats (STRs) is commonly used for human identity testing. Many commercial kits are available for this purpose, in which multiplex PCR for 10–16 STRs is usually used. For optimal results, the kits recommend rather limited conditions, which not all forensic samples can satisfy. We increased the efficiency of several commercial kits by modifying the components and reaction parameters (primer concentration and cycle number, and annealing and extension time). To simulate low copy number, we used 1:10 and 1:100 diluted samples compared to the recommended concentration. A change in cycle number, annealing and extension time, and primer concentration showed various results. Fine-tuning of PCR conditions by combining the described changes, decreasing the primer concentration, and increasing the annealing and extension time together with increasing the cycling number dramatically increased the efficiency of the reaction, even in low-copy-number simulated samples. Detailed information for several kit components or kits with different components targeting difficult sample conditions, including low copy number, would be of great help in forensics. Since the introduction of the so-called DNA fingerprint, namely, individual identification using DNA polymorphisms, much progress has been made (Butler et al. 1994; Krenke et al. 2002). The standard method is multiplex PCR amplification for several polymorphic short tandem repeats (STRs) using fluorescent-labeled PCR primers followed by electrophoresis. Given this background, commercially available kits from several companies have become popular. The AmpFlSTR Identifiler PCR Amplification Kit and the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems, Foster City, California) and the PowerPlex 16 System and PowerPlex Y System (Promega Corporation, Madison, Wisconsin) are representative of such kits. 1 Zhang AH, Seo SB, Yi JA, Kim HY and LEE SD