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Introduction to Forensic Science and Criminalistics Prepared by Peter Bilous Eastern Washington University Chapter 10
© 2007 The McGraw-Hill Companies, all rights reserved. DNA Analysis and Typing Genetics, Inheritance, Genetic Markers DNA – Nature and Functions Where DNA is Found in the Body – Nuclear (Genomic) and Mitochondrial DNA (mtDNA) Collection and Preservation of Biological Evidence Development and Methods of DNA Analysis Current DNA Typing Methods – Short Tandem Repeats (STRs) The Power of DNA to Individualize Biological Evidence Databasing and CODIS Applications of Forensic DNA Typing Newer DNA Technologies Strengths, Limitations, Promise, Hype
© 2007 The McGraw-Hill Companies, all rights reserved. I. Genetics, Inheritance, Genetic Markers Genetics is the science of inheritance The rules of inheritance were determined by Gregor Mendel in the late 1800s DNA is the genetic material of all living organisms, the chemical blueprint of life In higher animals, DNA is organized into structures known as chromosomes, found in the nucleus of cells Humans have 46 chromosomes
© 2007 The McGraw-Hill Companies, all rights reserved. I. Genetics, Inheritance, Genetic Markers DNA regulates cell activity by specifying how to make proteins Some proteins are structural, others function as enzymes Prior to DNA typing analysis, forensic scientists used proteins and enzymes as the genetic markers to try and individualize biological evidence Genetic differences among people that enable them to be distinguished are called genetic polymorphisms
© 2007 The McGraw-Hill Companies, all rights reserved. II. DNA – Nature and Functions The double stranded helical structure of DNA was elucidated in 1953 DNA is a polymer consisting of monomer units known as nucleotides Nucleotides consist of an organic base, a five carbon sugar (ribose) and phosphate Bases can be one of four compounds, abbreviated A, G, C, & T
© 2007 The McGraw-Hill Companies, all rights reserved. II. DNA – Nature and Functions The DNA molecule consists of two strands Each strand consists of a polymer of nucleotides, paired with the second strand by specific base pairings: A with T & G with C The strands are therefore complementary The position of the terminal phosphate residue designates the 5 end of a strand, with the other end being the 3 end
© 2007 The McGraw-Hill Companies, all rights reserved. II. DNA – Nature and Functions The entire complement of DNA in one cell is referred to as the genome Human DNA has ~3.5 billion base pairs (bp) The base sequence in the coding portions of the DNA is referred to as the genetic code The majority of DNA is non-coding and those regions contain tandemly repeated sequences that are important to the forensic analysis of biological evidence Tandem repeated sequences have core repeats that range from 2 bp to over 100 bp
© 2007 The McGraw-Hill Companies, all rights reserved. II. DNA – Nature and Functions The two DNA strands can be separated by heat or other conditions, a process referred to as melting or denaturation Strand separation is important for cells processes such as cell division, and for DNA analysis by PCR amplification DNA has two principal functions: replicating during cell division and coding for proteins The cell division process is called mitosis and involves the synthesis of new DNA (replication) catalyzed by enzymes called DNA polymerases
© 2007 The McGraw-Hill Companies, all rights reserved. DNA Replication
© 2007 The McGraw-Hill Companies, all rights reserved. II. DNA – Nature and Functions The DNA base sequence determines the chemical structure of all the proteins The genetic code is the sequence of bases in DNA, taken 3 at a time, that code for amino acids, the building blocks of protein DNA codes for proteins via an intermediate called mRNA The process of making mRNA from DNA is called transcription The process of making protein from mRNA is called translation
© 2007 The McGraw-Hill Companies, all rights reserved. Transcription and Translation
© 2007 The McGraw-Hill Companies, all rights reserved. III. Where DNA is Found in the Body Nuclear and Mitochondrial DNA Nuclear DNA or genomic DNA is found in the nucleus of cells Every cell in the human body has a complete identical copy of a persons DNA, with two exceptions: Mature red blood cells have no nuclear DNA Germ cells have only half of the genetic material (23 chromosomes)
© 2007 The McGraw-Hill Companies, all rights reserved. III. Where DNA is Found in the Body Nuclear and Mitochondrial DNA Mitochondria are structures within the cell that are responsible for making energy There are hundreds to thousands of mitochondria per cell Mitochondria have a small quantity of DNA known as mtDNA mtDNA is inherited only from ones mother Two regions of the mtDNA, HV1 and HV2, exhibit variations between individuals within the population and therefore can be of value in certain types of forensic cases
© 2007 The McGraw-Hill Companies, all rights reserved. IV. Collection and Preservation of Biological Evidence for DNA Typing Biological evidence should be thoroughly dried before packaging DNA tends to degrade in biological found in trace quantities or stains that are damp or warm or both Enzymes called DNases which degrade DNA, are released during putrefaction and are also present in some bacteria Substratum comparison specimens are important in situations of dilute biological samples or trace- type stains
© 2007 The McGraw-Hill Companies, all rights reserved. V. Development and Methods of DNA Analysis The foundations for forensic DNA typing analysis were laid down in the 1970s and 1980s by molecular biologists Most of the human DNA is non-coding, and a good portion consists of repetitive sequences A tandem repeat sequence consist of a sequence of bases that is repeated in a head-to-tail fashion numerous times Different individuals have a different number of repeat units at a particular locus These regions are called variable number of tandem repeat loci (VNTR) or minisatellites
© 2007 The McGraw-Hill Companies, all rights reserved. V. Development & Methods of DNA Analysis: Isolation (Extraction) of DNA DNA isolation involves the digestion of biological evidence with proteinase, an enzyme which breaks down proteins, releasing the contents of the cells After separating the DNA from other cellular components, tests are performed to quantify the DNA A variation known as the differential extraction procedure is used for mixtures of sperm cells and vaginal epithelial cells
© 2007 The McGraw-Hill Companies, all rights reserved. V. Development & Methods of DNA Analysis: The Beginning - RFLP The first forensic DNA procedure was known as restriction fragment length polymorphism (RFLP) analysis of VNTR loci It involved cutting DNA with enzymes known as restriction endonucleases, separating the fragments by electro- phoresis, transferring the fragments to a nylon membrane, and detecting alleles with DNA probes
© 2007 The McGraw-Hill Companies, all rights reserved. V. Development & Methods of DNA Analysis: The first PCR-Based DNA Typing Methods The polymerase chain reaction (PCR) is a copying process, yielding millions of copies of a defined segment of DNA The specificity is determined by small single stranded DNA molecules known as primers
© 2007 The McGraw-Hill Companies, all rights reserved. V. Development & Methods of DNA Analysis: The first PCR-Based DNA Typing Methods PCR based DNA Typing analysis has the following advantages over RFLP: PCR methods are many times faster PCR methods work with DNA that is degraded PCR methods work with much smaller amounts of DNA Cetus Corporation developed a PCR based technique in the late 1980s to detect alleles at the HLA-DQA1 locus Cetus Corp. later devised a Polymarker kit to detect alleles at 5 different genetic loci
© 2007 The McGraw-Hill Companies, all rights reserved. VI. Current DNA Typing Methods Short Tandem Repeats (STRs) The VNTR loci that form the basis of the current PCR-based DNA typing methods have repeat units of four or five base pairs These loci are called short tandem repeats (STRs) or microsatellites 13 STR loci were initially chosen for analysis as they provided a high level of individualization The choice of 13 STR loci was also based on the desire to have all forensic laboratories contribute profiles of the same genetic markers to the DNA database
© 2007 The McGraw-Hill Companies, all rights reserved. VII. The Power of DNA to Individualize Biological Evidence The level of individuality from DNA typing depends on the population genetics of the alleles found at the loci chosen for analysis Adding more and more loci to the DNA profile reduces the number of people who could possibly share it The frequency of alleles in the population at each of the loci tested are multiplied together to give an estimate of the probability of chance duplicate Probability estimates vary according to the ethnic/racial group
© 2007 The McGraw-Hill Companies, all rights reserved. VII. Databasing and CODIS There are some variations in the laws of individual States as to which offenders are DNA typed and stored Every state allows databasing of offenders convicted of sex crimes Forensic DNA profile databases have at least two parts: profiles from convicted offenders and profiles from biological evidence in unsolved cases There are also three levels of databases: national, state, and local
© 2007 The McGraw-Hill Companies, all rights reserved. VII. Databasing and CODIS The national file is called the Combined DNA Index System (CODIS) and is maintained by the FBI Many states and localities have their own databases, designated SDIS and LDIS, respectively Databases help to connect cases that may not otherwise be connected, and hits to a convicted offender tentatively identifies the depositor of the biological evidence
© 2007 The McGraw-Hill Companies, all rights reserved. VIII. Applications of Forensic DNA Typing The major applications of forensic DNA typing analysis are: criminal case civil cases identification of persons 1. Criminal Cases: Sexual assaults and blood transfer cases predominate DNA typing can include or exclude suspects DNA analysis is a way of linking or disassociating biological evidence from a person, not a mechanism for establishing guilt or innocence
© 2007 The McGraw-Hill Companies, all rights reserved. VIII. Applications of Forensic DNA Typing 2. Civil Cases: Disputed paternity cases predominate Typically done by clinical or other labs In rare sexual assault cases, parentage testing may support a criminal charge 3. Identification of Decedents: Mass disaster or criminal cases When traditional methods cannot be used Identification may involve direct comparison to a reference specimen or parentage testing
© 2007 The McGraw-Hill Companies, all rights reserved. IX. Newer DNA Technologies In sexual assault cases involving males who are azoospermic, the evidence consists of a mixture of male and female epithelial cells The standard DNA typing analysis approach is complicated and harder to interpret in these situations A number of polymorphic loci on the Y chromosome (Y STRs) can be typed in these types of cases to yield a male specific profile
© 2007 The McGraw-Hill Companies, all rights reserved. IX. Newer DNA Technologies Other types of variability in the human genome have possible forensic applications Single nucleotide polymorphisms (SNPs) result from single base differences from person-to- person at a particular location Thousands of SNPs are found in the human genome, but changing from STRs would require a significant effort Studies being conducted on animal and plant material have potential value to criminal investigations
© 2007 The McGraw-Hill Companies, all rights reserved. X. Strengths, Limitations, Promise, Hype Strengths: DNA technology is the most revolutionary tool available to the forensic scientists since fingerprints For the first time biological evidence can be effectively individualized Limitations: Not all crime scenes have biological evidence Mixtures of DNA are difficult to sort out Analysis of trace quantities of biological material is of questionable reliability Backlogs are significant
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