1. Chapter 10

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. DNA Replication

10. 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

11. Transcription and Translation

12. 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 person’s DNA, with two exceptions:
Mature red blood cells have no nuclear DNA
Germ cells have only half of the genetic material (23 chromosomes)

13. 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 one’s 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

14. 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

15. 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

16. 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

17. 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

18. 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”

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. X. Strengths, Limitations, Promise, Hype
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