Bruce R. McCord

A Study of PCR Inhibition Mechanisms Using Real Time PCR

Abstract:  In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co‐extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.

Capillary electrophoresis of polymerase chain reaction-amplified DNA using fluorescence detection with an intercalating dye

Abstract The application of the fluorescent intercalating dye, YO-PRO-1 to the analysis of polymerase chain reaction-generated DNA fragments from 120 to 400 base pairs in size was investigated. Analysis of samples was performed using a non-gel sieving buffer containing Tris—borate, hydroxyethyl cellulose, and the intercalating dye. Minimum detectable concentrations were less than 500 pg/ml DNA. For samples requiring resolution of four base pairs or less, it was necessary to add a second intercalating agent, ethidium bromide to the buffer. Using this procedure, a number of loci of interest in genetic typing were examined.

Explosive residue analysis by capillary electrophoresis and ion chromatography

Capillary electrophoresis is investigated for application as a complementary technique to ion chromatography in the analysis of low-explosive residues. Detection limits, interference problems, and matrix effects are examined by comparing the use of ion chromatography and capillary electrophoresis in parallel analyses. The residue from several different types of explosive devices are examined, and the results show capillary electrophoresis to be a useful new technique in explosive analysis yielding good sensitivity, high resolution and short analysis times.

Advances of capillary electrophoresis in clinical and forensic analysis (1999–2000)

In this paper, capillary electrophoresis in clinical and forensic analysis is reviewed on the basis of the literature of 1999, 2000 and the first papers in 2001. An overview of progress and relevant examples for each major field of application, namely (i) analysis of drug seizures, explosives residues, gunshot residues and inks, (ii) monitoring of drugs, endogenous small molecules and ions in biofluids and tissues, (iii) general screening for serum proteins and analysis of specific proteins (carbohydrate deficient transferrin, α1‐antitrypsin, lipoproteins and hemoglobins) in biological fluids, and (iv) analysis of nucleic acids and oligonucleotides in biological samples, including oligonucleotide therapeutics, are presented.

High Resolution Capillary Electrophoresis of Forensic DNA Using a Non-Gel Sieving Buffer

Abstract For the routine application of capillary electrophoresis to forensic DNA analysis, a number of requirements must be met. In the analysis of DNA amplified by the polymerase chain reaction these requirements include high resolution and consistent and reproducible runs. Genetic markers of interest in this study reproducible runs more base pairs long with repeat unnits as small as 2 base pairs. Normally gel-filled columns are necessary to resolve such small differences in size, however these columns suffer from a limited lifetime and a buildup of impurities over time. This paper reports an alternative method using a non-gel sieving buffer prepared from hydroxyethyl cellulose. Application of this method illustrates that a refillable, coated, open tubular column can achieve the necessary separation technique to a number of genetic markers of interest in forensic DNA analysis.

Detection of Smokeless Powder Residue on Pipe Bombs by Micellar Electrokinetic Capillary Electrophoresis

Improvised explosive devices are an increasing concern among law enforcement agencies within the United States because of their destructive capability. Capillary electrophoresis has been used previously for the forensic analysis of inorganic constituents in explosives. Micellar electrokinetic capillary electrophoresis (MECE), also known as micellar electrokinetic chromatography (MEKC), is well suited for the forensic analysis of organic constituents of these materials because of its high sensitivity and small sample requirements. In the present study, pipe bombs filled with known types of smokeless gun powder were detonated under controlled conditions. Samples of explosive residue were collected from the post-blast fragments and analyzed using MECE. The results were compared to the known types and analyzed to investigate the feasibility of matching post-blast residue to a specific powder used as explosive charge.

Forensic analysis of explosives using ion chromatographic methods

Abstract Applications of ion chromatographic analysis techniques including capillary electrophoresis and inverse photometric detection are explored in the analysis of low explosives and their residues. Past procedures for this anlaysis are reviewed and new techniques are introduced. A scheme for the investigation of trace evidence from bombing crime scenes using ion chromatographic techniques is developed. This scheme includes two sets of anionic and cationic analytical methods to allow identification and confirmation of the presence of inorganic explosive residue. Several different types of explosive devices are examined using this scheme, and the results are shown.

The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing.

The goal of this study is to explore the application of epigenetic markers in the identification of biofluids that are commonly found at the crime scene. A series of genetic loci were examined in order to define epigenetic markers that display differential methylation patterns between blood, saliva, semen, and epithelial tissue. Among the different loci tested, we have identified a panel of markers, C20orf117, ZC3H12D, BCAS4, and FGF7, that can be used in the determination of these four tissue types. Since methylation modifications occur at cytosine bases that are immediately followed by guanine bases (CpG sites), methylation levels were measured at CpG sites spanning each marker. Up to 11 samples of each tissue type were collected and subjected to bisulfite modification to convert unmethylated CpG‐associated cytosine bases to thymine bases. The bisulfite modified DNA was then amplified via nested PCR using a primer set of which one primer was biotin labeled. Biotinylated PCR products were in turn analyzed and the methylation level at each CpG site was quantitated by pyrosequencing. The percent methylation values at each CpG site were determined and averaged for each tissue type. The results indicated significant methylation differences between the tissue types. The methylation patterns at the ZC3H12D and FGF7 loci differentiated sperm from blood, saliva, and epithelial cells. The C20orf117 locus differentiated blood from sperm, saliva, and epithelial cells and saliva was differentiated from blood, sperm, and epithelial cells at a fourth locus, BCAS4. The results of this study demonstrate the applicability of epigenetic markers as a novel tool for the determination of biofluids using bisulfite modification and pyrosequencing.

A novel method for analysis of explosives residue by simultaneous detection of anions and cations via capillary zone electrophoresis

A novel electrolyte has been developed for the simultaneous separation of cations and anions in low explosive residue by capillary electrophoresis. This electrolyte contains 15mM alpha-hydroxyisobutyric acid (HIBA) as the buffer, 6mM imidazole as the cation chromophore, 3mM 1,3,6-naphthalenetrisulfonic acid (NTS) as the anion chromophore, 4mM 18-crown-6 ether as a cation selectivity modifier, and 5% (v/v) acetonitrile as an organic modifier. The pH was adjusted to 6.5 using tetramethylammonium hydroxide (TMAOH), an electroosmotic flow modifier. The method was optimized by varying the concentrations of alpha-HIBA, imidazole, and 1,3,6-NTS at three different pH values. The results provided a simultaneous indirect photometric analysis of both anions and cations with detection limits ranging from 0.5 to 5ppm for anions and from 10 to 15ppm for cations with a total run time of under 7min. The method was then applied to the analysis of Pyrodex((R)) RS and black powder, as well as several smokeless powders. The results obtained were consistent with previously reported results for separate anion and cation analysis and provide a faster, more complete analysis of each sample in a single chromatographic run.

Quantitation of polymerase chain reaction products by capillary electrophoresis using laser fluorescence.

In samples where the amount of DNA is limited, the polymerase chain reaction (PCR) can amplify specific regions of the DNA. A quantitative analysis of the PCR product would be desirable to ensure sufficient DNA is available for analysis. In this study, we examine the use of capillary electrophoresis (CE) with laser fluorescence detection for quantitation of PCR products. A coated open tubular capillary was used with a non-gel sieving buffer and a fluorescent intercalating dye to obtain results within 20 minutes. Using an internal standard, peak migration time was below 0.1% relative standard deviation (R.S.D.) with a peak area precision of 3% R.S.D. In comparison to quantitation by hybridization, (i.e., slot blot) and spectrophotometric analysis, capillary electrophoresis shows distinct advantages due to its ability to separate unincorporated primers and PCR byproducts from the targeted PCR product. The results demonstrate that CE can be used to monitor the quality and quantity of the PCR product.