Mechthild Prinz | PubMed Health

PubMed2006

The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA.

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.

DNA↗

PubMed2005

Validation of male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex.

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.

Animals↗

PubMed2005

Twelve short tandem repeat loci Y chromosome haplotypes: genetic analysis on populations residing in North America.

A total of 2443 male individuals, previously typed for the 13 CODIS STR loci, distributed across the five North American population groups African American, Asian, Caucasian, Hispanic, and Native American were typed for the Y-STR loci DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439 using the PowerPlex Y System. All population samples were highly polymorphic for the 12 Y-STR loci with the marker DYS385a/b being the most polymorphic across all sample populations. The Native American population groups demonstrated the lowest genetic diversity, most notably at the DYS393 and DYS437 loci. Almost all of the 12-locus haplotypes observed in the sample populations were represented only once in the database. Haplotype diversities were greater than 99.6% for the African Americans, Caucasians, Hispanics, and Asians. The Native Americans had the lowest haplotype diversities (Apaches, 97.0%; Navajo, 98.1%). Population substructure effects were greater for Y-haplotypes, compared with that for the autosomal loci. For the apportionment of variance for the 12 Y-STRs, the within sample population variation was the largest component (>98% for each major population group and approximately 97% in Native Americans), and the variance component contributed by the major population groups was less than the individual component, but much greater than among sample populations within a major group (11.79% versus 1.02% for African Americans/Caucasians/Hispanics and 15.35% versus 1.25% for all five major populations). When each major population is analyzed individually, the R(ST) values were low but showed significant among group heterogeneity. In 692 confirmed father-son pairs, 14 mutation events were observed with the average rate of 1.57x10(-3)/locus/generation (a 95% confidence bound of 0.83x10(-3) to 2.69x10(-3)). Since the Y-STR loci reside on the non-recombining region of the Y chromosome, the counting method is one approach suggested for conveying an estimate of the rarity of the Y-haplotype. Because the Y-STR loci are not all in disequilibrium to the same extent, the counting method is a very conservative approach. The data also support that autosomal STR frequencies can be multiplied by the upper bound frequency estimate of a Y-haplotype in the individual population group or those pooled into major population groups (i.e., Caucasian, African American, Hispanic, and Asian). These analyses support use of the haplotype population data for estimating Y-STR profile frequencies for populations residing in North America.

Canada↗

PubMed2005

Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex.

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.

Animals↗

PubMed2005

Forensic applications of laser capture microdissection: use in DNA-based parentage testing and platform validation.

AIM: To report on the successful use of Laser Capture Microdissection (LCM) as a tool for isolation of human chorionic villi from admixed maternal tissue. Subsequent DNA isolation for forensic short tandem repeat (STR) analysis for parentage testing was performed in two cases of alleged sexual assault of female victims. We also performed validation of the LCM instrument platform, using archival formalin-fixed human fetal products of conception (POC), for which microdissection was utilized to separate maternal (decidua) and fetal (chorionic villus) components. METHODS: To isolate DNA from placental chorionic villi admixed with maternal decidua recovered after spontaneous or therapeutic abortion, LCM was used to separate fetal from maternal cells. In contrast to the relatively crude conventional microdissection performed using a narrow pipette, needle, or scalpel blade, LCM allows cell- or tissue-specific isolation of placental chorionic villi from archival paraffin-embedded tissue sections, leaving the maternal tissue intact. RESULTS: After polymerase chain reaction (PCR) amplification of villi after LCM of 9-15 STR loci, the quantity and quality of DNA yielded from fetal cells isolated by LCM was sufficient for PCR analysis and successful forensic parentage testing. The validation data obtained on two sets of formalin-fixed archival POC tissues from anonymous donors demonstrated the encouraging reproducibility of these protocols and procedures. CONCLUSION: We demonstrated the reliability and utility of LCM for forensic applications when high specificity of a particular analyzed cell population or tissue is required. Care must be taken during routine pathology procedures to avoid contamination of tissues with admixture of extraneous DNA.

Adolescent↗

PubMed2005

Optimization of a simple, automatable extraction method to recover sufficient DNA from low copy number DNA samples for generation of short tandem repeat profiles.

AIM: To develop an automated, high throughput extraction protocol in order to produce database eligible profiles from fingerprints and other low copy number (LCN) DNA sources. METHODS: Extraction of either purified control DNA or buccal cells, for example, with commercial kits was compared to extraction with a simple digestion buffer and a subsequent concentration and purification. Results were evaluated based on the amount of DNA recovered and the completeness of the DNA profiles produced. RESULTS: Simple procedures with fewer steps were superior to commercial kits, such as DNA IQ (Promega, Madison, WI, USA) and QiaAmp (Qiagen, Valencia, CA, USA), and other protocols with many manipulations. The optimized protocol included a thirty-minute incubation with 0.01% SDS and proteinase K at 56 degrees C, followed by an incubation at 100 degrees C for 10 minutes. Concentration of the extract and removal of the SDS was accomplished with a Microcon 100 (Millipore, Bedford, MA, USA), which can be assembled into a 96 well plate, the Microcon-96 Retentate Assembly Plate (Millipore) for automation. The addition of 1 ng Poly A RNA to the Microcon significantly improved DNA recovery. CONCLUSION: A one-step sample digestion followed by sample concentration/purification minimized sample loss and maximized amplification input. Moreover, this methodology can be easily adapted for automation. Implementation of this protocol, due to the numerous potential sources of LCN DNA samples, will enhance the recovery of biological evidence from crime scenes and may be a source of database profiles.

Automation↗

PubMed2004

Evaluation of an automated liquid hybridization method for DNA quantitation.

The AluQuant (Promega Corporation) liquid hybridization DNA quantitation method was evaluated on an automated robotic platform (Biomek 2000, Beckman Coulter, Fullerton, CA) for use in forensic PCR-STR systems. DNA from bloodstains and buccal swabs was extracted by three different methods: Chelex, Qiagen and DNA IQ (Promega). Samples were quantitated using both the Quantiblot and the AluQuant systems. Concordance between methods was determined by comparing the average AluQuant DNA concentrations for samples having matching (binned) Quantiblot values. Studies testing the "accuracy" (STR analysis), precision, sensitivity, and specifies specificity of the AluQuant method were also conducted. The effect of inhibitors (carpet, denim, and suede) was evaluated. The results indicate that the AluQuant quantitation system equals the Quantiblot system in "accuracy", sensitivity, precision, and primate-specificity. While extracts from denim and suede affected (inhibited) both systems minimally, the carpet extracts produced a sharp increase in DNA quantitation values in the AluQuant but not the Quantiblot system. The speed and user-friendliness of the AluQuant system on a robotic platform offer specific advantages to the forensic community.

Animals↗

PubMed2004

A comparison study of hair examination methodologies.

A study was conducted to investigate the accuracy between two methods of hair analysis: PCR-STR DNA analysis and microscopic comparison analysis. Standard sets of pubic hairs were collected from volunteers, and unknown sets were generated from these samples. Three out of five (60%) of the hairs analyzed produced full DNA profiles that were correctly matched to the standard sets. DNA analysis was inconclusive (partial or no DNA profile) for two out of five (40%) of the samples. In contrast, the microscopic comparison analysis correctly matched four out of five (80%) of the samples to the standard sets but mis-identified one out of five (20%) of the samples. These results reinforce the practice of preliminary microscopic hair examination in narrowing down a set of hairs for DNA analysis. Microscopic comparison analysis is sufficiently reliable to remain a rapid and inexpensive method for forensic hair analysis.

DNA↗

PubMed2003

Y chromosome STR haplotypes and the genetic structure of U.S. populations of African, European, and Hispanic ancestry.

To investigate geographic structure within U.S. ethnic populations, we analyzed 1705 haplotypes on the basis of 9 short tandem repeat (STR) loci on the Y-chromosome from 9-11 groups each of African-Americans, European-Americans, and Hispanics. There were no significant differences in the distribution of Y-STR haplotypes among African-American groups, whereas European-American and Hispanic groups did exhibit significant geographic heterogeneity. However, the significant heterogeneity resulted from one sample; removal of that sample in each case eliminated the significant heterogeneity. Multidimensional scaling analysis of R(ST) values indicated that African-American groups formed a distinct cluster, whereas there was some intermingling of European-American and Hispanic groups. MtDNA data exist for many of these same groups; estimates of the European-American genetic contribution to the African-American gene pool were 27.5%-33.6% for the Y-STR haplotypes and 9%-15.4% for the mtDNA types. The lack of significant geographic heterogeneity among Y-STR and mtDNA haplotypes in U.S ethnic groups means that forensic DNA databases do not need to be constructed for separate geographic regions of the U.S. Moreover, absence of significant geographic heterogeneity for these two loci means that regional variation in disease susceptibility within ethnic groups is more likely to reflect cultural/environmental factors, rather than any underlying genetic heterogeneity.

Africa↗

PubMed2003

Characterization of a novel dimorphism in the 5' flanking region of the short tandem repeat (STR) locus, c-fes/fps (FES).

The FES short tandem repeat (STR) locus contains seven to 14 repeats of the tetranucleotide sequence ATTT. A novel 10 base pair dimorphism in the 5' flanking region of the FES locus was characterized in four broad populations: African-American, Hispanic, Caucasian, and Asian. The absence of the 10 base pair sequence, or (-) allele, was closely linked to FES STR alleles with 10 or fewer repeats. The presence of the 10 base pair sequence, or (+) allele, was closely linked to FES STR alleles with 12 or more repeats. The (-) and (+) alleles occurred equally often in FES STR allele 11. The nucleotide sequence (5'-GGCTGTTTTG-3') of the (+) allele, located 179 base pairs upstream of the FES STR, was determined to be consistent within and among the four populations. Statistical and sequence analysis confirmed the linkage between the two polymorphic sites. The results indicate that the exclusion rate of the FES locus is increased, above that for the STR alone, when both polymorphic characteristics are considered.

5' Flanking Region↗

PubMed2002

Online Y-chromosomal short tandem repeat haplotype reference database (YHRD) for U.S. populations.

We describe here an online Y-chromosomal short tandem repeat haplotype reference database (YHRD) for U.S. populations, which represents 9-locus Y-STR haplotypes for 1705 African-Americans, European-Americans and Hispanics as of October 2001. This database is available online (http://www.ystr. org/usa/), free to access and was generated in order to supply the U.S. forensic DNA community with a valuable resource for frequencies of complete or incomplete 9-locus Y-STR haplotypes, as well as information about typing protocols and population genetic analyses. Pairwise R(ST)-statistics derived from the Y-STR haplotypes indicate no significant substructure among African-American populations from different regions of the U.S., nor (usually) among European-American and Hispanic populations. Thus, pooling of Y-STR haplotype data from regional populations within these three major groups is appropriate in order to obtain larger sample sizes. However, pooling of different major populations is generally not recommended due to statistically significant differences between African-American populations and all European-American/Hispanic populations, as well as between some European-American and Hispanic populations.

Black People↗

PubMed2002

Typing Y chromosome STR haplotypes using redesigned primers.

A panel of Y-specific STR loci, including DYS19, DYS389, DYS390, DYS391, DYS392, and DYS393 was analyzed using horizontal nondenaturing polyacrylamide gel electrophoresis with a discontinuous buffer system (horizontal disk-PAGE). In order to obtain correct results for the larger DYS389 and DYS392 alleles, it was necessary to design new primers that bind closer to the repeat region and lead to a significant reduction of the amplified fragment size. Using the modified primer sets the horizontal disk-PAGE results were consistent with a nondenaturing approach using fluorescent primers and a 377 automated sequencer. The modified procedure also amplifies the second repeat stretch at the duplicated DYS389 locus as a single fragment, which results in an immediate allele identification. The results indicate that horizontal disk-PAGE with silverstaining is a simple approach to type the recommended Y-specific STR markers.

DNA Primers↗