1. DNA Fingerprinting

2. Sources of Biological Evidence
Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Tissue

3. Characteristics of DNA
DNA is a polymer with repeating units called nucleotides.
Each nucleotide has:
sugar molecule
phosphate group
nitrogen containing group (base)

4. Structure of DNA
Sugar and phosphate form the BACKBONE and the bases fall into the center.
String of NUCLEOTIDES form each strand of DNA.

6. DNA is a double stranded helix
Two DNA strands come together to form a DNA molecule.
Watson and Crick credited for discovery of the DNA double helix (Rosalind Franklin shunned!!)

7. Selective base pairing holds the strands together
Adenine (A) only pairs with Thymine (T)
Cytosine (C) only pairs with Guanine (G)

8. Complementary Base Pairing
Bases can occur in any sequence on one strand. Opposite bases will be on the other strand.
A T C C T G G C T T A T C G C
T A G G A C C G A A T A G C G
Explain BASE PAIRS
Average chromosome (where DNA is organized ) contains 100 million base pairs
Only 4 bases but infinite number of combinations…..think of 4 letters of alphabet and the number of words produced.
The order of the letters defines the role and function of the DNA ….ie….what proteins are produced etc.

9. Where do proteins come from?
Proteins are made up of units called amino acids
Order of the amino acids determines the function and shape of the protein.
Triplet or three bases  amino acid
C-G-T Alanine
C-T-A Aspartate
The genetic information that determines the order of the amino acids is in the sequence of the DNA……………..the DNA sequence for alanine –aspartate would be CGTCTA!

10. Normal and sickle cell red blood cells.

11. Genetic diseases e.g. Sickle cell anemia: mutation in hemoglobin.
Normal sequence: C C T G A G G A G
Sickle cell anemia: C C T G T G G A G
Changes the amino acid from
glutamine  valine

12. Human Genome Project Cost: $450 million (1990-2003)
Aim: determine the order/sequence of bases on DNA contained in all 23 chromosomes.
Why?

13. DNA is “supercoiled”!

14. Replication of DNA

15. Replication of DNA Parent strand must unwind.
Nucleotides are assembled to make new “daughter” strand.
Process continues until entire parent strand is copied.
End up with two exact copies.
Think of each strand as a positive and a negative copy (like a photograph film). Each strand has the same information.
Base pairing rules apply.
DNA polymerase 1. make sure the bases are in the cortrect order
2. “proof read” the sequence to make sure that they are correct.

16. Individual nucleotides
DNA in the Cell
chromosome
cell nucleus
Double stranded DNA molecule
Individual nucleotides
Target Region for PCR

17. Polymerase Chain Reaction
DNA (e.g. crime scene, victim, suspect)
+ DNA polymerase
+ nucleotides (A, T, C, G)
+ primers
Place in Thermal Cycler
 multiple, exact copy of original DNA
DNA can replicated OUTSIDE a living cell too!!!! If we add the correct components….this is the basis of PCR.
Technique allows small pieces of DNA found at a crime scene to be amplified millions of times in a couple of hours.
Machine is called DNA THERMAL CYCLER>goes through a series of heating and cooling cycles automatically. Each cycle doubles the amount of DNA.
So sample size is no longer a limitation.

18. PCR Copies DNA Exponentially through Multiple Thermal Cycles
Original DNA target region
Thermal cycle
Thermal cycle
Thermal cycle
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

19. Polymerase Chain Reaction (PCR).

20. Restriction enzymes
Restriction enzymes: “cut” DNA at specific sequences.
e.g. Eco R1, Hind III, Pst I etc.
EcoRI SmaI
“sticky ends” “blunt ends”
Once a particular DNA site has been identified as “coding” for a particular protein………one can “cut and paste” pieces of DNA together and put them inside of cells.
The cell will then make that particular protein and copy it into the daughter cells every time it divides.
This is RECOMBINANT DNA TECHNOLOGY//genetic engineering
Plasmids are usually from bacteria.

21. Recombinant DNA Technology
Plasmid: vehicle used to transfer the “new” DNA into any cell.
Each new generation of bacteria/cell will carry the new piece of DNA. Each cell will then make the protein dictated by that particular DNA sequence.
E.g. produce insulin or human growth hormone.

22. DNA Typing
Not all of DNA sequences code for the production of proteins
Tandem repeats can distinguish individuals.
Concern here with NUCLEAR DNA talk about mitochondrial DNA later,
Not all letter sequences in the DNA code for proteins………some are random sequences that are repeated throughout the DNA many times……these are nonsense sequence called TANDEM repeats.
Tandem repeats located through DNA Typing………as with any genetic trait …these are inherited from the parents…….one chromosome from one parent and the other from the other parent.

23. To identify the tandem repeats we use DNA Typing………
To identify the tandem repeats we use DNA Typing……….next slide shows the process

25. HLA DQ alpha system (DQA 1)
First commercial and validated PCR-based genetic marker system.
DNA typing of hair, saliva, semen stains etc.
DQA 1 – used for forensic science and accepted in court.
Originally used for tissue typing.
DQA1 typing of hair, saliva on envelopes, stamps, cigarette butts, semen stains//
advantage over RFLP DNA typing…….only need 1/50th amount of DNA (1 billionth of a gram) ……..can yield useful information from degraded samples that sometimes fail RFLP Typing
……show figure and revisit after talking about PCR.
Highlight probes are on the membrane Not the same as RFLP DNA typing.

27. PCR produces multiple identical copies.

28. PCR animation (http://users.ugent.be/~avierstr/principles/pcrani.html)
How does PCR work?
Consider, -G-C-T- T-C-C-A-G-
-C-G-A-A-G-G-T-C-
Identify “primer sequences” and design primers.
Add DNA + primers + nucleotides (G,A,T,C) + DNA polymerase.
Heat DNA (separate the strands)
Cool DNA (primers anneal and DNA polymerase assembles new strand)
1 CYCLE = two complete identical copies of DNA
PCR animation (
Heating cycle to 92 degrees and cooling cycle is 72 degrees.
Number of cycles usually cycles, with DNA amount doubling each cycle.
Thus amplification of a small amount of DNA……..revisit the HLA DQ alpha system overhead.

29. Short Tandem Repeats (STRs)
locations (loci)on the chromosome that have sequence elements that repeat themselves within the DNA molecule.
3-7 bases in length, repeated many times.
Every person has 2 STR types for each element…..one inherited from each parent.
Analysis requires STR’s must be identified, number of repeats defined and sequence of bases flanking the STR
Latest method of DNA typing…………..STR analysis
. The more STRs one can characterize …..the smaller the percentage of the popoulation from which they can emanate…….this gives rise to the concept of MULTIPLEXING……….that is one can simultaneously extract and amplify a combination of different STRs NEXT SLIDE
Locations on the chromosome that contain short sequences of 3-7 bases “sequence elements”//found in great abundance//less susceptible to degradation.
Sequences are known and many have been named….THO1
Advantages………many hundreds of STRs can be examined to increase the probability of individualization//figure in textbook shows the greater the # of STR characterized…the smaller the frequency of occurrence in the general population (probability value determined).
DNA Typing (RFLP) or PCR related (such as HLA DQ alpha or STR analysis) is an essential investigative tool.

30. STR Analysis and Multiplexing
1. Extract STR* (eg. THO1,ie. A-A-T-G) from biological sample.
2. Amplify by PCR
3. Separate on electrophoresis gel
4. Examine the distance moved to estimate to determine the number of STR repeats that exist.
*Multiplexing allows simultaneous extraction and amplification of a number of different STRs
MULTIPLEXING
…….this gives rise to the concept of MULTIPLEXING……….that is one can simultaneously extract and amplify a combination of different STRs
Even identified Y-STR markers allowing a “mixture of blood, saliva, vaginal fluid to proove if multiple males are involved in an assault.

31. Multiplexing PCR Over 10 Markers Can Be Copied at Once
Sensitivities to levels less than 1 ng of DNA
Ability to Handle Mixtures and Degraded Samples
Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

32. Federal DNA Identification Act
1994
Combined DNA Index System (CODIS): facilitate the exchange of DNA typing data among police agencies investigating violent crimes and sexual assaults.
// authorizes the FBI to national DNA identification system for law enforcement
FBI policy for accepting evidence for DNA testing is explained in APPENDIX III of textbook
TABLE 13-1 lists the thirteen CODIS STRS and their probabilities of identity.

33. DNA Index System Mandated collection of :
Collection of DNA samples from convicted offenders
Establishment of DNA databases for law enforcement.
All 50 states have mandated collection and establishment of a database.
Controversial???……..topic for class discussion.

35. Mitochondrial DNA.
Nuclear DNA (both parents) but mitochondrial DNA (maternal lineage only).
mtDNA is circular
mtDNA only contains information for 37 genes.
2 regions (HV1 and HV2) have greatest number of variances.
Significantly more sensitive than nuclear DNA typing BUT more costly, time consuming and rigorous.
Mitochondria are “power plants of our cells 90% of energy our body needs to function.
Each cell has mitochondria (lots of DNA!!)

36. Collection and Preservation of Biological Evidence for DNA Analysis
Photograph, notes, sketches
Assume all body fluids are infectious
Look for blood in less obvious places.
Packaging!
Refrigerate and store away from sunlight.
DNA control samples.
Hypervariable region 1 and 2 are compared in mt DNA typing.//
Charred remains (insufficient DNA) then there may still be enough mitochondrial DNA for analysis. OR missing or deceased person when retrieve maternal DNA for comparison purposes BUT all individuals with same maternal lineage will be indistinguishable.
mt DNA typing does not yet exceed the power of STR analysis