Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.

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The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?

Biological Basis for DNA Evidence, Peter Gill and John Buckleton Historical and Background Biology Understanding PCR Profiles A Framework for Interpreting Evidence, John Buckleton The Frequentist Approach The Logical Approach The Full Bayesian Approach A Possible Solution A Comparison of the Different Approaches Population Genetic Models, John Buckleton Product Rule Simulation Testing Discussion of the Product Rule and the Subpopulation Model A Complex Case Example - DNA Evidence and Orethral James Simpson Relatedness, John Buckleton and Christopher Triggs Conditional Probabilities Joint Probabilities The Unifying Formula The Effect of Linkage Validating Databases, John Buckleton Which Is the Relevant Population? Population Databases Validating the Population Genetic Model Estimating Q Descriptive Statistics for Databases Sampling Effects, John Buckleton and James Curran Bounds and a Level Methods for Assessing Sampling Uncertainty Minimum Allele Probabilities Discussion of the Appropriateness of Sampling Uncertainty Estimates Mixtures, Tim Clayton and John Buckleton Frequentist Approaches Bayesian Approaches Statistical Evaluation of Mixtures Low Copy Number, John Buckleton and Peter Gill Changes in LCN Profile Morphology The Interpretation of LCN Profiles Non-autosomal Forensic Markers, Simon Walsh, SallyAnn Harbison, and John Buckleton Forensic Mitochondrial DNA Typing Forensic Y Chromosome Analysis Forensic X Chromosome Analysis A Famous Case Example - The Romanovs Parentage Testing, John Buckleton, Tim Clayton, and Chris Triggs Evaluation Of Evidence Paternity Trios: Mother, Child and Alleged Father Non-autosomal DNA Use of the Sub-Population Model of Balding and Nichols to Evaluate the Paternity Index Relatedness in Paternity Cases Multiple Children Inconsistencies in the Mendelian Pattern 'Exclusions' Paternity Trios: Mother, Child and Alleged Father Considering the Possibility of Silent (Null) Alleles Disaster Victim Identification, Identification of Missing Persons, and Immigration Cases, John Buckleton, Chris Triggs, and Tim Clayton Mitochondrial or Nuclear DNA? Human Remains - Obtaining a Profile from Bodily Remains Extraction of DNA from Bone, Tooth, Hair and Nail Complicating Factors DNA Intelligence Databases, Simon Walsh and John Buckleton A Brief History Functional Aspects Legislation Aspects of Forensic Significance Social and ethical considerations Interpretation Issues Associated with DNA Databases

Forensic DNA Evidence Interpretation

Fundamentals of Forensic DNA Typing , Fundamentals of Forensic DNA Typing , کتابخانه دیجیتال جندی شاپور اهواز

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Fundamentals of Forensic DNA Typing

Low copy number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. There are a number of methodologies to increase sensitivity of detection to enable LCN typing. These approaches encompass modifications during the polymerase chain reaction (PCR) and/or post-PCR manipulations. Regardless of the manipulations, when processing a small number of starting templates during the PCR exaggerated stochastic sampling effects will occur. The result is that several phenomena can occur: a substantial imbalance of 2 alleles at a given heterozygous locus, allelic dropout, or increased stutter. With increased sensitivity of detection there is a concomitant increased risk of contamination. Recently, a commission reviewed LCN typing and found it to be "robust" and "fit for purpose." Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing. The findings of the commission seem inconsistent with the nature of LCN typing. While LCN typing is appropriate for identification of missing persons and human remains and for developing investigative leads, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. A more in-depth evaluation by the greater scientific community is warranted. The issues to consider include: training and education, evidence handling and collection procedures, the application or purpose for which the LCN result will be used, the reliability of current LCN methods, replicate analyses, interpretation and uncertainty, report writing, validation requirements, and alternate methodologies for better performance.

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Validity of low copy number typing and applications to forensic science

PCR-based DNA typing of biological evidence is now widely used in forensic analyses due to the obvious advantages of enhanced sensitivity, the ability to distinguish discrete alleles and efficacy with degraded samples. A multiplex short tandem repeat (STR) system has been previously developed which successfully co-amplifies six STR loci HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA) in conjunction with the X-Y homologous gene Amelogenin. This is known as the second generation multiplex system (SGM). Detection of the PCR products is undertaken on ABD 373A or 377 automated sequencers using denaturing polyacrylamide gels coupled with fluorescent-based technology. We have evaluated this system for routine forensic use and demonstrated that the technique is robust and reproducible under conditions consistent with those encountered in a forensic environment. A total of 132 stains from simulated and actual casework were analysed, together with relevant control areas and reference samples. The success rate was high with 76% of stains giving full profiles; we were also able to successfully detect and interpret mixtures. No mistyping was observed. A detailed examination of each of these profiles has assisted in the development of guidelines for casework interpretation. Although artefacts, stutter peaks and undenatured DNA were occasionally observed, these did not interfere with the accuracy of interpretation. In addition 38 samples, previously examined using the quadruplex system, were analysed with the SGM to enable a direct comparison to be made between the systems. The performance of the system with poor quality samples demonstrated its use as a rapid and powerful technique for individual identification.

The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies.

Improvements in detection limits/sensitivity and lower sample consumption are potential benefits of reducing PCR reaction volumes used in forensic DNA typing of crime scene samples. This premise was studied first with experimental mixtures and a nine-loci megaplex, which demonstrated stochiometric amplification and accurate detection. Next, adjudicated casework samples were subjected to amplification under 15 different template DNA to PCR reaction volume ratios. Reduction of PCR reaction volume and DNA down to 10 microL and 0.500 ng, respectively, produced identical profiles with the same signal intensity and heterozygous allele peak height ratio (HR). Reduction to 5 microL and 0.063 ng yielded HR values that were slightly affected in one to three STR loci. PCR reaction volume reduction can enhance detection and sensitivity while reducing the consumption of irreplaceable crime scene samples.

STR DNA typing: increased sensitivity and efficient sample consumption using reduced PCR reaction volumes.

Historically, formalin fixed (FF) tissues could not be used as a source of DNA in forensic science due to the fact that the DNA was too degraded for DNA analysis. With the introduction of the polymerase chain reaction (PCR) technique to forensic science, the usefulness of DNA from this biological material has been re-evaluated. This study evaluates the potential use of DNA from FF and formalin fixed paraffin embedded (FFPE) tissues in 13 PCR systems; HLA DQα, LDLR, GYPA, HBGG, D7S8, GC, D1S80, vWA31, THO1, F13A1, FES/FPS, TPOX, and CSF1PO. The first six, HLA DQα, LDLR, GYPA, HBGG, D7S8, and GC are reverse dot blot systems, D1S80 is an amplified fragment length polymorphism (AmpFlp) system and the others are short tandem repeats (STRs). This study shows that FFPE tissue which has not been fixed in formalin for more than three days is a useful source of DNA for 12 of the 13 PCR systems. In contrast, FF tissue did not prove to be a reliable source of DNA for the PCR techniques examined here.

The Applicability of Formalin-Fixed and Formalin Fixed Paraffin Embedded Tissues in Forensic DNA Analysis

A system of four short tandem repeat loci (HUMVWF31A, HUMTH01, HUMF13A1, and HUMFES/FPS) has been tested in co-amplification with forensic (post-mortem and post-coital) DNA samples. Semiautomated DNA typing was employed to analyze polymerase chain reaction (PCR) products formed by extension of primers labeled with a fluorescent dye at the 5'-terminus. Most DNA extracts could be typed, although a few required the addition of bovine serum albumin or a pretreatment by ultrafiltration in order to obtain sufficient signal for typing. Balanced signals for the alleles were obtained frequently across the loci, although preferential amplifications of HUMTH01 was observed often with the forensic samples. Band splitting due to nontemplate nucleotide addition to the blunt ends of the amplimers was frequently detected for the DNA extracted from the forensic samples. A data-base was constructed for the African-American population and compared with a Caucasian database. Few differences were observed across the two populations, except at the locus HUMTH01. The fluorescence-based system facilitates large-scale databasing, because the PCR products run off the gel, allowing more than one set of samples to be analyzed per run. Polyacrylamide gel reuse did not diminish genotyping accuracy.

Forensic applications of a rapid, sensitive, and precise multiplex analysis of the four short tandem repeat loci HUMVWF31/A, HUMTH01, HUMF13A1, and HUMFES/FPS

Ann C. Juston

Papers

STR DNA typing: increased sensitivity and efficient sample consumption using reduced PCR reaction volumes.

The Applicability of Formalin-Fixed and Formalin Fixed Paraffin Embedded Tissues in Forensic DNA Analysis

Forensic applications of a rapid, sensitive, and precise multiplex analysis of the four short tandem repeat loci HUMVWF31/A, HUMTH01, HUMF13A1, and HUMFES/FPS