1. The Indispensable Forensic Science Tool
DNA:
The Indispensable Forensic Science Tool

2. APPLICATIONS Colin Pitch Fork, or the OJ Simpson trial
FORENSICS - TO LINK A SUSPECT TO A CRIME SCENE AND FOR CRIMMINAL EXONERATIONS
Colin Pitch Fork, or the OJ Simpson trial
TO IDENTIFY A MISSING OR DEAD PERSON
ID Saddam Hussein and his sons,
or when we find Osama Bin Ladin
TO LINK A SUSPECT TO AN EVENT
Clinton – Lewinsky Scandal

3. Why is DNA an Indispensable Tool Forensic Science?
Study into the structure of the human genome has led to the discovery that portions of the DNA structure of certain genes are as unique to each individual as fingerprints

4. What is DNA?
Each of our 60 trillion cells contains nuclear genetic material called chromosomes
Arranged on these chromosomes are genes
The gene is the fundamental unit of heredity
Instructs the body cells to make proteins that determine everything from hair color to our susceptibility to diseases.
Each gene is composed of DNA coded with a specific sequence designed to carry out a specific function.

5. What is DNA? Polymer – repeating monomers of nucleotides.
DNA nucleotides are composed of a deoxyribose sugar molecule, a phosphorous-containing group, and a nitrogen-containing molecule called a base.

6. James Watson & Francis Crick
1953 discovered the structure of Deoxyribonucleic Acid – DNA
Double Helix
Composed of 4 different type bases
complementary base pairing of Adenine always with Thymine and Guanine with Cytosine.
No restriction on how many bases are to be sequenced on a strand of DNA.

7. Nitrogenous bases are always paired A-T and C-G
Held together by weak hydrogen bonds

8. The backbone of the DNA chain is supported by the phosphorous and sugar groups – forming the rungs of the “ladder”

9. A T A G C C A A C C A A T A T C G G T T G G T T
Replication of a single strand of DNA
A T A G C C A A C C A A
T A T C G G T T G G T T

10. DNA at Work DNA controls the production of Proteins
Proteins are polymers of amino acids
There are 20 known amino acids yet thousands of different proteins can be produced performing different functions.
The amino acids act like the letters in the alphabet (26 letters in different combinations = thousands of words) so 20 different amino acids in different sequences equal about 100,000 different proteins each with its own unique shape which determines its function.

11. How can the DNA sequence dictate the sequence for a particular protein?
The template DNA is transcribed in the nucleus by RNA (Ribonucleic Acid)
Thymine on the DNA nucleotide is replaced by Uracil on the RNA nucleotide so A is paired with U.
Each amino acid is attached to a triplet complementary codon of a RNA transcribed DNA sequence.

12. A T A G C C A A C C A A U A U C G G U U G G U U
So, a DNA Sequence of:
DNA template Strand
A T A G C C A A C C A A
U A U C G G U U G G U U
U A U C G G U U G G U U
Would have an mRNA sequence of:
RNA Strand of Transcribed DNA
Each group of three nucleotide bases, an RNA triplet codon, is read in order to determine which amino acid is placed in the sequence. The sequence determines the type of protein that is produced.
Use the following table to determine which of the 20 amino acids would make up this sequence.

13. U A U
C G G
U U G
G U U

14. mRNA Sequence: U A U C G G U U G G U U Leucine Arginine Valine
Would result in this protein amino acid sequence
Tyrosine
Tyrosine
Arginine
Leucine
Valine

15. U A U C G G U U G G U U U A U C G G U U C G U U
a one base change in the mRNA Sequence:
U A U C G G U U G G U U
U A U C G G U U C G U U
Phenylalaine
Arginine
Valine
Would result in this protein amino acid sequence
Tyrosine
Tyrosine
Arginine
Phenylalaine
Valine

16. Does all my DNA code for proteins?
99.9% of all human DNA is the same
Exons – makes up about 5% of human DNA
Codes for Proteins
Introns – makes up about 95% of human DNA.
Filler or “junk” portion of the DNA sequence.
Variaitions within intron DNA accounts for the .1% difference between each of us and is used in DNA fingerprinting.

17. EARLY DNA FINGERPRINTING TECHNIQUE
Restriction Fragment Length Polymorphisms – RFLP “RiFLiPs”
1980 – Ray White Describes first Polymorphic RFLP Genetic Marker.
DNA FROM SEVERAL INDIVIDUALS WAS DIGESTED WITH THE RESTRICTION ENZYME, SEPARATED BY GEL ELECTROPHORESIS AND DETECTED BY SOUTHERN BLOT ANALYSIS USING A RADIOACTIVE PROBE FOR THE D14S1 LOCUS ON CHROMOSOME 1. DIFFERENT BANDING PATTERNS WERE SEEN FOR DIFFERENT INDIVIDUALS.
DNA FROM SEVERAL INDIVIDUALS WAS DIGESTED WITH RESTRICTION ENZYME AND ANALYZED VIA A SOUTHERN GEL BLOT ANALYSIS USING RADIOACTIVE PROBE FOR THE D1451 LOCUS ON CHROMOSOME 1.
DIFFERENT BANDING PATTERSN WERE SEEN FOR DIFFERENT INDIVIDUALS.
DNA FROM SEVERAL INDIVIDUALS WAS DIGESTED WITH THE RESTRICTION ENZYME ECOR1, SEPARATED BY AGOROSE GEL ELECTROPHORESIS AND DETECTED BY SOUTHERN BLOT ANALYSIS USING A RADIOACTIVE PROBE FOR THE D14S1 LOCUS ON CHROMOSOME 1, DIFFERENT BANDING PATTERNS WERE SEEN FOR DIFFERENT INDIVIDUALS.
The restriction fragment length polymorphism or RFLP (sometimes called DNA fingerprinting or profiling) is considered to be the more accurate of the two. This test examined sequences of base pairs in a section of a DNA strand with a high probability of being entirely unique to the donor. When a match is found, there is no question that the donor was at the scene of the crime. It’s very conclusive and finalizing. Unfortunately, the RFLP requires many sample cells from the crime scene like several strands of hair or large splatters of blood. The cells have to be “fresh”, too--that is, undamaged and recently dead. The test takes anywhere from 3 weeks to three months to complete, also. Although the PCR test, or polymerase chain reaction test, isn’t quite as accurate, it takes much less time to complete--a week at most. The test can be performed with minute crime scene samples, to, which helps investigators who have little physical evidence. The DNA doesn’t have to be recently collected, either; the PCR test can still be performed even years after--decades after--the fact, and still be just as accurate. This is because the PCT method involves copying the available DNA and analyzing only one specific gene, oftentimes the gene called HLA DQ alpha. Geneticists look for certain types of the gene (alleles) to determine matches. If no match is found, the donor was not at the crime scene. Matches are not conclusive, though, because many people may have--and are more than likely to have--the same alleles as the donor. Still, juries have convicted suspects based on PCR test results.

18. Restriction Fragment Length Polymorphism
DNA from several individual was digested (cut) with a restriction enzyme.
Detected by Southern Blot analysis.
Different banding patterns were seen for different individuals. 1984
Sir Alec Jeffreys developed and used multi-locus RFLPs to catch Colin Pitchfork.

19. RFLP/ Southern blot analysis
Gel with an uncut and cut samples of DNA. Note that the sizes of the cut DNA fragments add up to the size of the uncut DNA.
This variation in the noncoding region can nevertheless be detected by RFLP analysis because the substitutions can result in the production or elimination of a restriction enzyme recognition site. This in turn results in different sized fragments produced from the DNA of different individuals.
Sir Alec Jefferies worked with RFLPs in one of the first cases using DNA evidence to convict a rapist and murder and exonerate an emotionally disturbed man individual in 1987 using RFLPs

20. Large
fragments
small
fragments
Size of the fragments cut as a result of the restriction enzyme will influence the distance the fragments will travel within the gel during electrophoresis. Smaller fragments will travel further than large fragments.

21. RFLPS/SOUTHERN GEL DISADVANTAGES
It is expensive, time, and labor intensive with a low throughput.
One locus is analyzed at a time.
Less sensitive – Requires a large sample size.
It is limited in obtaining DNA from a crime scene since you need enough blood, hair, or semen.
It requires whole genome analysis.
Needs relatively recently collected samples as the DNA tends to degrade.
It is time and labor intensive, low throughput. One gene analyzed at a time.
RFLP method requires a relatively large amount of DNA along with radioactive labeling visualized on a gel. To expose an X-ray film with a radioactive labeled probe takes about a week. You have to do that for each system, so the entire process can take many weeks
Less sensitive---Requires ample, high-quality specimen.
The RFLP requires many sample cells from the crime scene like several strands of hair or large splatters of blood. It is limited in obtaining DNA samples from crime scene since you need large amounts of blood, hair or semen It may require a court order to obtain a blood sample from a suspect.
You can only look at one locus at a time in a gel to get a clear picture and link on individual to another
Multilocus RFLPs banding patterns are not as clear in linking the suspect who did the crime individual to a crime scene.
Sir Alec Jefferies worked with RFLPs in one of the first cases using DNA evidence to convict a rapist and murder and exonerate an emotionally disturbed man individual in 1987 using RFLPs
RFLP analysis requires whole cell genomes. Why? Because you are not amplifying a locus as in PCR. Here you are looking at a locus insitu
PCR techniques offer the advantage of requiring only trace degraded or amounts of DNA and they can be done overnight

22. Variable Number Tandem Repeats - VNTRs
More than 30% of the human genome contains repetitive DNA nucleotide sequences.
These repeat sequences or tandem repeats vary in number between individuals and can be inherited from both parents
Variable Number Tandem Repeats - VNTRs
15 to 35 nucleotide bases per repeat
Up to 1000 repeats
Results in DNA segments of thousands of bases in length
Short Tandem Repeats - STRs
1-7 nucleotide bases per repeat
Results in DNA segments of about 400 bases in length
Shorter segments make it less susceptible to degredation
The human genome contains many repetitive DNA sequences that is considered satellite DNA consisting of nucleotide repeats of as little as and up to ~ 100 nucleotides long.
They are of no know function
SATELLITE DNA WAS FIRST DISCOVERED THOUGH ULTRACENTRAFUGATION WHICH PRODUCED SATELLITE DNA BANDS IN TEST TUBES
ANALYSIS REVEALED THEY CONSISTED OF REPEATED DNA SEQUENCES
THESE DNA BANDS WERE CALLED “SATELLITE DNA” AND CREATED VARIOUS BANDING PATTERNS BASED ON THEIR COMPOSITION.
THESE SATELLITE BANDS WERE ANALYZED USING GEL ELCETRPHORESIS AND DNA CONSISTING OF REPEATED SEQUENCES IN THE SAME ORIENTATION WERE DISCOVERED
SATELLITE DNA SEQUENCES ARE REPEATED IN TANDEM ARRAYS OF MANY THOUSANDS OF COPIES, HENCE THEY REASSOCIATE READILY WITH COMPLEMENTARY DNA PROBES for DNA assays
Satellite DNAs are usually in heterochromatic regions of the chromosomes, often around centromeres and near telomeres. They are not transcribed into RNA.
"Satellite" actually refers to any DNA population whose density differs from that of the bulk of the DNA.
Satellite DNAs consist of short sequences repeated many times, in the same orientation, and without intervening spacer sequences.
Sequences arranged in this manner are tandem repeats.
Not all tandemly repeated DNAs are density gradient satellites because some, by chance, have the same density as the bulk DNA. Some density gradient satellites are organelle DNA. Yet, the term "satellite DNA" is usually used as a synonym for tandemly highly repeated DNA.
Repeated sequences may be interspersed with other repeated sequences
Regions of DNA where short nucleotide sequences repeat over and over; regions of tandem repeats
MINISATELLITES OR VARIABLE NO. TANDEM REPEATS VNTRsDS nucleotides in a repeat unit
14-40 possible repeats in an allele
- 10 to 100 bp per repeat
copies per loci
- Many thousands of loci
LOCATION
Often found concentrated in centromeres and sprinkled throughout the genome
MICROSATELLITES – Discovered in 1989
SHORT OR SIMPLE TANDEM REPEATS (STRs)
1-8 bp repeat
5-100 copies per loci – many loci
Randomly dispersed though out the genome
These STR loci are found on almost every chromosome in the genome.
VNTRs and STRs are hypervariable between individuals, and are the basis of DNA fingerprinting

23. VNTRs with RFLPs SOUTHERN GEL ANALYSIS
By digesting genomic DNA with restriction enzymes that cuts outside of these repeats it is possible to obtain different multiples of repeats that can be detected by hybridizing a DNA probe that detects (hybridizes to) the single repeat.
Number of repeats influences the length of the cut fragments after exposure to the restriction enzymes

24. DNA FINGERPRINT EXAMPLE VNTR WITH SINGLE LOCUS
DID CHILD 4 DEVELOP A NEW ALLELE FOR 7 REPEATS?
ADOPTED CHILD F M
POSSIBLE CHILDREN
REPEAT
NUMBER 5 8
IS LANE 4 FROM AN UNRELATED PERSON WITH A SIMILAR ALLELE TO THE MOTHER? 6
the DNA fingerprint isBased on just 2 VNTR alleles, ie. a single unique locus.. A single unique locus, while useful for paternity testing, is not unique and has limited applications for populations (DNA profiling) because 2 individuals can have the same allele by chance. The VNTR sequences in man are clearly related and by choosing 2 other VNTR probes, you can detect more varients between individuals( 33.6 and 33.15,) each probe now detects up to 60 variants.
Though typically each probe detects 17 different variants.
STRs are now also used for DNA fingerprinting. 3
ANAYLZE MORE LOCI SIMULTANEOUSLY 2

25. VNTR/RFLP MULTILOCUS DNA FINGERPRINTING
Some VNTR sequence segments are found at only a single locus in the human genome. Probes made of these sequences are single locus probes and yield this pattern when used to probe RFLP blots of DNAs
Other VNTR sequence segments occur at many loci in the human genome. These loci are dispersed among the chromosomes. Multi-locus VNTR probes yield patterns like bar codes
YOU STILL NEED THE WHOLE GENOME FOR
RFLP ANALYSIS
Such probes are called multi-locus probes (MLPs), because they detect many hypervariable regions simultaneously. These probes typically detect between 15 and 20 variable DNA fragments larger than 3.5 kb per individual, plus many smaller bands that are not used in mapping or fingerprinting casework. DNA fingerprinting can be based on the use of these MLPs.
Produces a pattern resembling a bar code. A number of bands appear because the probe uses is repetitive sequence homologous to a number of loci dispersed though out the genome
Some VNTR sequence segments are found at only a single locus in the human genome. Probes made of these sequences are single locus probes and yield patterns such as those at left when used to probe RFLP blots of DNAs of six individuals.
Other VNTR sequence segments occur at many loci in the human genome. These loci are dispersed among the chromosomes. Multi-locus VNTR probes yield patterns such as those at right.

26. VNTRs/ RFLPS SOUTHERN GEL ANALYSIS
One of the drawbacks of VNTRs with Southern gel analysis was that they required relatively large amounts of DNA and was very time consuming
PCR changed everything due to the ability to amplify a locus in a short period of time
VNTRs because of their size, they are usually detected by digestion of the genomic DNA by restriction enzymes followed by hybridization with a locus-specifc radioactive probe just as with RFLPs

27. Short Tandem Repeats (STRs) PCR Analysis
AATG
THE FORENSIC COMMUNITY SETTLED ON
TETRANUCLEOTIDE REPEAT
Advantages of PCR-based STRs Analysis
Increased sensitivity - RFLP analysis requires nanograms of DNA. PCR requires ng of DNA and is fast
The quantity and integrity of the DNA sample is less of an issue with STR-PCR-based typing methods than with conventional RFLP methods.
The forensic DNA community has moved primarily towards tetranucleotide repeats, which may be amplified using PCR with greater fidelity than dinucleotide repeats.
FOR DNA SEQUENCE VARIABILITY TO BE USEFUL TO IDENTIFY INDIVIDUALS
Simple to analyze
Provide data that can uniquely identify individuals and can withstand critical examination in court
Able to identify degraded DNA in poorly preserved biological samples (found in crime scenes)
Have a fast throughput
STR – PCR DNA fingerprinting fulfills these 4 criteria
the repeat region is variable between samples while the flanking regions where PCR primers bind are constant
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another

29. What is PCR? Polymerase Chain Reaction
A technique used to amplify (copy or multiply) DNA
Can be done with minute amounts of DNA
Uses DNA’s natural replication by DNA polymerase
DNA is heated (denatured) to separate the strands of DNA
primers (short strands of DNA target specific regions of DNA for replication) hybridize (anneal) with the separated strands of DNA
DNA polymerase directs the rebuilding of the double-stranded DNA molecule using a mixture of free nucleotides
PCR animation
PCR Tutorial

33. Uses of STR and RFLP
RFLPs have provided valuable information in many areas of biology, including:
screening human DNA for the presence of potentially deleterious genes

34. providing evidence to establish the innocence of,
or a probability of the guilt of, a crime suspect by DNA "fingerprinting“

35. Paternity
DNA fingerprinting can be used to identify a child’s parents. Each child inherits one set of chromosomes from each parent.
Some RFLPs are inherited from the mother and some from the father.

36. Interpret the results of the following DNA fingerprints
Both daughter 1 and son 1 share RFLPs with both the mom and dad, while daughter 2 has RFLPs of the mom but not the dad, and son 2 does not have RFLPs from either parent.

37. The police use the same analysis to determine the identity of a person at a crime scene. After collecting DNA samples from the scene and any suspects, the police amplify and digest the DNA with a restriction enzyme. The samples are run on an agarose gel, and the bands found at the crime scene are aligned with those of the suspects’.

38. The DNA fingerprint from suspect 1 matches up with the fingerprint of the sperm DNA from the crime scene. You can also see that the female cells from the scene match the victim’s DNA.

39. Capillary Electrophoresis
The preferred technique of characterization of STRs
Preformed in a thin glass column (coated with a gel polymer) immersed in a reservoir of buffer liquid with platinum coated high-voltage electrodes
DNA sample solution is injected into one end of the column.
DNA will migrate under the influence of an electrical potential at a speed that is related to the STR fragment lengths.
The opposite side of the column is connected to a laser beam detector that tracks the separated STRs as they emerge from the column.
This is sensitive enough to detect fragments that are different by only 1-4 bases in length.

43. Profiler Plus Allelic Ladders
D3S1358
VWA
FGA
Profiler Plus Allelic Ladders
AMEL
D8S1179
D18S51
D21S11
D5S818
D13S317
D7S820

44. COfiler Allelic Ladders
D3S1358
D16S539
TH01
TPOX
CSF1PO
AMEL
D7S820
COfiler Allelic Ladders

45. What are Y-STRs and how are they useful to the forensic scientist?
Short tandem repeats located on the human Y chromosome
Originates only from a male donor of DNA
20 different Y-STR markers
Running a Y-STR profile is useful when multiple males are involved in a sexual assault.
Yields only one band or peak for each STR type
Simplifies the appearance and interpretation of the DNA profile

47. What is mitochondrial DNA and how is it used in forensic analysis?
Mitochondria are the site of cellular respiration in all our cells (energy producers).
Many mitochondria are found in the cytoplasm of each cell.
Can self replicate – has its own looped DNA
mtDNA
Inherited only from the mother
mtDNA analysis is more sensitive then nuclear DNA but is more rigorous, time consuming, and costly.

48. What is mitochondrial DNA and how is it used in forensic analysis?
Two regions of mtDNA have been found to be highly variable in the human population – HV1 and HV2. Analysis requires:
PCR
Sequencing the hyper-variable regions
FBI maintains a database containing the base sequences from HV1 and HV2 – Currently contains about 5,000 sequences which can be used to determine how common or rare a specific sequence is compared to the current database.
Not as discriminated as nuclear DNA analysis
Best used when there is no nuclear DNA available for testing

49. What is CODIS ? CODIS is an acronym for Combined DNA Index System
Essentially administered by the FBI – provides CODIS training and software

50. CODIS Alleles 13 CODIS Alleles Amelogenin Gene – determines gender
STRs of tetrameric repeats
Amelogenin Gene – determines gender

51. DNA Profile Frequency Calculations Genotype Probability at any STR Locus
Forensic DNA analysis requires population databases for the STR loci evaluated.
Probability calculations are based on knowing allele frequencies for each STR locus for a representative human population.
Allele frequency – the number of copies of the allele in a population divided by the sum of all alleles in a population.

52. DNA Profile Frequency Calculations Genotype Probability at any STR Locus
For a heterozygous individual, if the two alleles have frequencies of p and q in a population, the probability (P) of an individual of having both alleles at a single locus is
P = 2pq
If an individual is homozygous for an allele with a frequency of p, the probability (P) of the genotype is
P = p2.
Individual A has the genotype 15, 18 at the locus D3S1358. In a reference database of 200 U.S. Caucasians, the frequency of the alleles 15 and 18 was and , respectively. The frequency of the 15, 18 genotype is therefore
P = 2 (0.2825) (0.1450) = .0819, or 8.2%.

53. MULTIPEX STR SYSTEM
The simultaneous extraction and amplification (copying) of a combination of different STRs
The variety of alleles present in a population is such that a high degree of discrimination among individuals in the population may be obtained when multiple STR loci are examined.

54. Since the CODIS Loci are predominantly on separate chromosomes (only 2 are found on chromosome 5 at opposite ends), it insures that the probability of any two bands being inherited together is the product of individual occurrences i.e. independent assortment.
This aspect strengthens the significance of probability of occurrence for court cases.

55. Probability for a DNA profile of Multiple Loci
If databases of allele frequency for different loci can be shown to be independently inherited by appropriate statistical tests, the probability for the combined genotype can be determined by the multiplication (product rule).
The probability (P) for a DNA profile is the product of the probability (P1, P2, ... Pn) for each individual locus, i.e.
Profile Probability = (P1) (P2) ... (Pn)
When all 13 CODIS STR markers are included in the DNA profile, the probability can result in extremely low numbers.
1.3 X (1016), or no more frequent than 1 in 7.7 quadrillion individuals (7.7 million billion), which is more than a million times the population of the planet.

56. Steps in DNA Sample Processing
Sample Obtained from Crime Scene or Paternity Investigation
Biology
DNA
Extraction
Quantitation
PCR Amplification
of Multiple STR markers
Technology
Separation and Detection of PCR Products
(STR Alleles)
Sample Genotype Determination
Genetics
Comparison of Sample Genotype to Other Sample Results
If match occurs, comparison of DNA profile to population databases
Generation of Case Report with Probability of Random Match

57. Allele Frequencies
STRBase

58. The
End
THESE SATELLITE BANDS WERE ANALYZED USING GEL ELCETRPHORESIS AND DNA CONSISTING OF REPEATED SEQUENCES IN THE SAME ORIENTATION WERE DISCOVERED
SATELLITE DNA SEQUENCES ARE REPEATED IN TANDEM ARRAYS OF MANY THOUSANDS OF COPIES, HENCE THEY REASSOCIATE READILY WITH COMPLEMENTARY DNA PROBES for DNA assays
Satellite DNAs are usually in heterochromatic regions of the chromosomes, often around centromeres and near telomeres. They are not transcribed into RNA.
"Satellite" actually refers to any DNA population whose density differs from that of the bulk of the DNA.
Satellite DNAs consist of short sequences repeated many times, in the same orientation, and without intervening spacer sequences.
Sequences arranged in this manner are tandem repeats.
Not all tandemly repeated DNAs are density gradient satellites because some, by chance, have the same density as the bulk DNA. Some density gradient satellites are organelle DNA. Yet, the term "satellite DNA" is usually used as a synonym for tandemly highly repeated DNA.
Repeated sequences may be interspersed with other repeated sequences
Regions of DNA where short nucleotide sequences repeat over and over; regions of tandem repeats