1. JS 113: Introduction to DNA Typing
Pre class activities
Announcements and Assignments
Exam 2 Return- Avg 68.1 Range 39-86
II. Learning Objectives
Understand why scientists study DNA. Applications of forensic DNA
Introduction to DNA- Definition and Inheritance review
DNA structure
Be able to draw DNA structure – Base Pairing AT, GC
Understand DNA replication
Summary of Introduction to DNA
d. Overview of Methods used in Forensic DNA typing
Screening
Extraction
Quantification
e Be able to define: cell, nucleus, chromosome,
alleles, homozygous vs heterozygous
Be able to draw a Punnett Square to illustrate allele inheritance

2. Announcements, Assignments and Schedules
Announcements and Assignments
Study for quiz
Extra Credit – 1 point each – due Monday 19 Nov
Read Esslinger et al Using STR analysis to detect human DNA from exploded pipe bomb devices. JFS 49(3) I will provide hard copies to those interested today.
Provide 500 word summary with 3Q and 3A by Monday
Schedule reminders- All completed notebooks due last class
Pizza party next weds for thanksgiving- we will have an in class quiz (open book team quiz for all those that attend)

3. Top 10 ways you can tell you’re a forensic DNA scientist
10. You have your kid’s DNA types vs. their pictures on your wall.
9. When your kids were born, you got an extra newborn footstick smpl.
8. When your kids get injured you are more interested in collecting the blood than dressing the cut.
7. You have a backlog of fingernails, hair, and teeth from every member of your willing lineage awaiting testing.
6. You want to open a paternity service for the Jerry Springer show.
5. When you stay in hotels you bring a portable UV light so you can avoid unsanitary latent stains, and then you collect them.
4. You know forensic DNA takes longer than a CSI commercial break.
You see stains where no one else can.
You spend your day looking at dirty underwear.
1. Your license plate reads “ OJ DID IT!”
* Butler, J ACS Meeting presentation

4. Who Cares? WE ALL DO! Law Enforcement
Criminal Investigation- Casework, Databanks
Reuniting immigrant families- Paternity
Missing persons
Evolutionary, Agricultural and Zoological applications
Assessing genetic diversity
Fingerprinting endangered species and pathogens
Assessing unrelatedness to breed for increasing genetic diversity
Assessing relationships for all biological predictions
Ancient DNA analyses for reconstructing history (how we populated the globe)
Other Human Applications
Making sense of the Human Genome project results- Bioinformatics
Developing rapid medical diagnostics such as those associated with triplet repeat diseases (STRs)- (Moxon et al Sci Amer. 280:94)
Understanding the molecular basis of development, disease and aging
Screening candidates for bone marrow/organ transplants and grafts
WE ALL DO!

5. Report on the DNA Evidence Backlog and the national DNA Database (CODIS) -2004
… 542,700 criminal cases with biological evidence are awaiting DNA testing… includes 52,000 homicide and 169,000 rape cases…not being tested due to lack of resources, funding… and the current law
…information from United Kingdom officials that show DNA evidence at property crime scenes increased suspect identification by 44 percent.
Investigation of property crimes has significantly increased the conviction rate of rapists and murderers in conjunction with the database… Proposition 69 passed in CA- Nov 2004 leading to the expansion of the CA State DNA Database- Increase in forensic DNA jobs and funding.
The national DNA Database contains an offender index 2,643,409 and a forensic index 119,782 records 27,806 investigations aided

6. President’s DNA Initiative Justice for All act of 2004 www.dna.gov
1 billion dollars in funding over 5 years
From to address the backlog and to assist in developing more discriminating, more cost effective, and faster methods of DNA typing
$98 million just announced

7. Butler, J. 2005 ACS Meeting presentation

8. Applications of Forensic DNA Technology
Criminal Casework: Solving or linking crimes
Accident and Mass disasters: Determine the source of human remains (Hurricane Katrina).
Military conflicts: Missing in action, tomb of the unknown soldier.
Biodefense: Detecting class A-C pathogens
Missing Person Cases Paternity Testing
Missing children
Innocence Project: Exonerating the innocent

9. Innocence Project http://www.innocenceproject.org/
Innocence Project founded at the Cardozo Law School in New York - spearheaded the plight of the imprisoned innocent.
Scheck and Neufeld utilize volunteer law students and attorneys to review hundreds of cases of people who say they have been falsely convicted, usually of rape or murder, and, when appropriate, arrange for DNA tests that may support their claim of innocence.
More than 150 innocent prisoners have been exonerated with new DNA tests and evidence which excluded them as participants in the crimes for which they had been convicted.

10. Where is it? How is it stored? DNA is found in every
DNA Facts and Jargon
Where is it? How is it stored?
DNA is found in every
*cell= basic unit of life
Inside nuclei (organization center for the cell) and mitochondria (ATP powerhouse of the cell) & chloroplasts for plants- (making our food via photosynthesis)
Nuclei are not found in red blood cells
In white blood cells, saliva, skin, hair fingernails, urine, feces, vomitus, earwax etc.

11. Individual nucleotides
DNA in the Cell In nuclei, mitochondria and chloroplasts (plants) organized in chromosomes (wound around histones) “DNA double bagging”
chromosome
cell nucleus
Double stranded DNA molecule
Individual nucleotides
Target Region for PCR

12. I. Intro to DNA : Facts and Jargon DNA: Deoxyribonucleic acid
DNA: Deoxyribonucleic acid
Different in every *individual
The same in every **cell of an individual's body
*except for identical twins that have the same DNA - "The time honored method of cloning humans"
** diseased individuals may be mosaics

13. DNA function What’s it do? DeoxyriboNucleic Acid : blueprints of life
Replication, Information Storage and Mutation
Central Dogma
information flow >
DNA >RNA------>protein
transcription translation

14. DNA Organization and Inheritance Human Genome Contains 23 Pairs of Chromosomes It is inherited from your mom and dad
X Y
Sex-chromosomes

15. Basic Chromosome Structure and Nomenclaturep
(short arm)
centromere
telomere q
(long arm)
Band 5
Band 3
Chromosome 12
12p3
12q5
Chromosome 12
P= short arm
Q= long arm
Center= Centromere (involved in mitosis and meiosis attachment to spindle fibers)
End= Telomere (involved in aging)
D12S459
D= DNA
12= Chromosome 12
S=Single copy sequence
459= 459th locus described on C12

16. Definitions of Locus and Allele
2 pairs of Homologous chromosomes (white from dad, dark from mom)
Locus (singular) or Loci (plural) are defined locations where specific genes or markers are found
Alleles are different forms of the same gene or marker
When alleles have the same form on a locus they are said to be homozygous. When different they are heterozygous
Card analogy- locus is King- alleles are the 4 suits

17. Review- Mendelian Genetics
Law of Independent Segregation-
Big D and little d will evenly segregate into the next generation
And results in equal inheritance from mom and dad
Name Genotype Gametes possible
Steve D,D D
Julie d,d d D
d d
Dd Dd
Punnett Square

18. Inheritance Review
Dad is homozygous (A, A) and mom is heterozygous (A, a).
In your teams, draw the Punnett Sqaure that demonstrates the inheritance of these alleles.
What percentage of their children will be heterozygous?

19. Review- Mendelian Genetics Law of Random Assortment
This law states that markers on different chromosomes are generally inherited independently of one another and are not inherited together more often than might be expected by chance.
Those displaying random assortment are said to be in linkage equilibrium.
Those that show genetic linkage such as those located close together on the same chromosome are said to be in linkage disequilibrium and are found more often together than expected by chance. Example is blonde hair and blue eyes.
Markers that are in linkage equilibrium are desirable for forensic DNA. The product rule can be applied to those displaying random assortment thus resulting in a higher power of discrimination than those that are linked.

20. Phosphate-sugar backbone
DNA Structure
What is it?
Bases (AGCT) form the stairs of the ladder, are faithfully paired and exhibit differences. P
S-A : T-S
P P
S-G : C-S
 Sugars (S) and phosphates (P) form the sides of the ladder (identical for all DNA).
Bases (AGCT) form the stairs of the ladder, are faithfully paired by hydrogen bonds and exhibit differences. A : T and G : C
A = T
G  C
T = A
C  G T C A G 5’ 3’
denatured
strands
hybridized
Hydrogen bonds
Phosphate-sugar backbone

21. DNA Structure
Primary genetic material is composed of two complementary strands
Form a double helix or twisted ladder
Sides are sugar phosphate and the steps are base pairs
Four Bases- 2 Purines – Adenine and Guanine and 2 Pyrimidines- Cytosine and Thymine
Asian Guys are Pure!

22. DNA Structure Nucleotides are the building blocks themselves composed of PBS
Nucleotides-PBS
Phosphate (negative charge)
Base (AGCT-Asian Guys Can Teach)
Sugar (deoxyribose-5C)
Phosphate-Sugars
Connected by phosphodiester linkages

23. Basic Components of Nucleic Acids
Phosphate
Sugar
Base
5’end |
Phosphate
Sugar—Base…
3’end

24. DNA Structure 2 Complimentary, Antiparallel Strands held together by Base Pairs- H Bonds.
A:T held with 2 H Bonds
G:C held with 3 H Bonds

25. Where’s Daddy? 11 14 Father 12 14 Child #1 8 14 Child #2 11 12
PCR product size (bp) 11 14
Father 12 14
Child #1 8 14
Child #2 11 12
Child #3 8 12
Mother

26. DNA St. Patricks Day Salute to the Molecule of Heredity From Biology 110- UNC 1993 Steve Lee
The molecular structure today
Is heredity’s DNA
With nucleotides
completely comprised
of a sugar and phosphate and base
The bases you see are so keen
They include thymine and adenine
Cytosine and one more
with guanine can store
all the info with rungs in between
The sides of the ladder you know,
are sugar and phosphate which show
that Franklin was right
double helix is tight
ten base pairs per turn in a row
Adenine and thymine can base pair
Forming two hydrogen bonds for one stair
Cytosine and guanine
pair with three in between
and are equal in size when compared
DNA strands are just not the same
One is coding and one is called lame (anticoding)
They are opposite
in direction and this
is called antiparallel in name
Complimentary nature of strands
lets replication proceed just as planned
with A paring to T
and G pairing to C
the fidelity is precise and quite grand

27. Summary 1 Why study DNA DNA Biology and Genetics
Law enforcement, evolution, agricultural, and human applications-medical diagnostics
DNA Biology and Genetics
DNA is contained in cells –the basic unit of life
Found in nuclei, mitochondria and chloroplasts
Organized in chromosomes. Located at positions called loci and come in different forms or alleles.
Homozygous if the same, heterozygous if different
Alleles segregate independently and assort randomly when on different chromosomes. Random assortment is desired for forensic DNA loci.
DNA Function and Structure
DeoxyriboNucleic Acid : blueprints of life
Replication, Information storage and mutation RIM
Central Dogma
DNA >RNA------>protein
transcription translation

28. Summary 2 DNA Structure and Function continued: DNA Replication
Bases of DNA are Adenine, Guanine, Cytosine and Thymine- Asian Guys Can Teach: AGCT
Base pairing is A to T and G to C- DNA is where its AT
Sequence of Bases Store information- Like the sequence of numbers in a Phone Number
Nucleotides are the building blocks (dNTPs) themselves made of phosphate base and sugar= PBS- The only station Sierra and Gabriel can watch
DNA base pairs- DNA velcro (David Letterman
DNA Replication
Semi-conservative- Half republican (old) /half democrat (new)
Template directed with base pairing (AT, GC)
5 required ingredients- primer, template, Mg, dntps, DNA polymerase (PTMDD)

29. Steps to Sample Processing
Screening- The Art- Presumptive and confirmatory tests- blood, semen and saliva
DNA Extraction- Many types
organic phenol/chloroform
Chelex
FTA paper
Silica based extractions
DNA Quantitation (yield gels, slot blot, real time PCR)
PCR Amplification
Separation/Detection
Genotype Determination
Interpretation- Report Writing- Court testimony

30. Steps in Forensic DNA typing
(Figure 6.1 Rudin and Inman 2001)
Evaluation- Is it there?
1. Start with biological sample
2. Screen- blood? Semen? Saliva, human?
Extraction- Get and clean DNA
3. Open cells  Get DNA
4. Methods to get DNA and purify DNA
Quantify- Determine quality and quantity?
5. Quantify-
How good and how much did you get?
Type to determine and compare alleles
6. RFLP vs PCR
7. Determine alleles and compare DNA types
Or alleles present in samples and references
Interpretation of Results

31. Review: DNA is organized inside the cell nucleus and mitochondria

32. DNA Extraction
After screening tests are performed, a spot of the material containing the biological sample is cut and placed into a tube.
In one type of extraction method (organic), heat and chemicals are added, and protein is removed.
Then the pure DNA is recovered by filtration in which the non-DNA material goes through a sieve.
(analogous to a collection of your pasta in a colander)

33. Differential Extraction Method For Sexual Assault Evidence
Female
cell
Spermatozoa
Isolation of DNA from mixtures of cells in sexual assault evidence
Based on differences in cell membranes
Spermatozoa membranes have special cross links (sulphur-sulphur bonds)
These membranes are quite resistant to opening.
Vaginal epithelial cells do not contain these membranes and are more easily broken open
Sperm and v cell
mixture
Lysis- open
v cell extract
Female DNA
Female DNA
Lysis- open
sperm extract
Male DNA
Male DNA

34. Quantification of DNA
Following extraction, the next step is to determine the quantity of the DNA
DNA typing methods RFLP and PCR require different amounts and different quality of DNA.
RFLP typically required 50ng. PCR typically requires less than 0.5ng to 1 ng: 100 times less!

35. Quantification of DNA using Gel Electrophoresis
(-)
Total DNA can be quantified by running the samples in a gel.
Typically, gels are made up of agarose (a carbohydrate from seaweed).
Known DNA quantities are included
Samples are then subject to an electric current and is called electrophoresis.
DNA is negatively charged and will migrate toward the positive electrode…
Comparisons of the results are done visually or with computer software to determine the amount of DNA in the unknown sample.
L K K – u u u
wells
Intact
DNA
Degraded
DNA
(+)
Direction of DNA fragment movement
Smaller fragments move faster and are found
Near the bottom of the gel

36. Slot Blot quantification : DNA-DNA Hybridization DNA is where it’s AT
DNA samples may contain non human DNA
In order to quantify the amount of human DNA in a sample, a human specific test is required
One such test is DNA-DNA hybridization using a human specific probe: D17Z1

37. Slot blot hybridization
Like in yield gels, known amounts of DNA (human) are included
DNA hybridization of D17Z1 will occur only if the sample contains human DNA
Detection of the hybridized fragments is done using an enzyme linked assay- yielding light or color

38. Comparison of Methods used for DNA Quantification
Method Ease Cost Sensitivity Result
UV Spectrophotometry Total DNA
Yield Gel electrophoresis Int vs deg DNA
Slot Blot Human DNA
Yield Gel blot Int. vs. deg human DNA Pico-green microtitre plate Total DNA
Alu Quant Human DNA
Real time PCR assays Human DNA

39. DNA Methods 1) Extract 2) Quantitate 3) Distinguish Size Content
Restriction Fragment Length Polymorphisms (RFLP)
Polymerase Chain Reaction (PCR)

40. The base sequence can exhibit differences in length and content between individuals.
Dr. Dre AAAGAAAGAAGAAAC...
DMX AAAGAAAGAAGA...
OutKast AAAGAAAGAAGT...
SnoopDogg ... AAAGAAAGAAGA...
Britney Spears ... AAAGAAAGAA...
Christina Aguilera ... AAAGAAAGAT...
Eminim AAAGAAAGC...
NSYNC AAAGAAAGT...
Boyz to Men ... AAAGAAAG…
Although different between individuals* DNA is identical in every cell of an individuals body** Some exceptions*identical twins**diseased individuals, mtDNA (sport analogs)

43. RFLP Role Playing 1. Students are assigned a base.
2. Sequence is provided with a restriction site 5’GGCC3’ at the end.
3. A person acts as the restriction enzyme and cuts the strand.
4. The gel (classroom) is loaded, power supply is turned on (lights) and fragments are asked to slink through the class toward the front (+) end.
5. Power is stopped and we visualize the difference in migration of the short and long fragment

44. Polymerase Chain Reaction:
PCR is simply repeated rounds of DNA replication
PCR based systems are rapid, require less material than RFLP and less time for typing
Molecular xeroxing
Calvin and Hobbes example

45. DNA Polymerase catalyzes the template directed (A-T, G-C), incorporation of dNTPs (PP is released) forming a 3’-5’ phosphodiester linkage
Direction of synthesis 5’3’ using primer 3’OH to attach incoming nucleotide
5-P’
Primer
3-OH’
3-OH’
dNTP
P-P- OH
Template
Mg++
DNA polymerase
5-P’

46. PCR is simply repeated rounds of DNA replication
5’ 3’
3’ 5’
Template- DNA from blood etc.
Step 1: Denature
Separate H bonds with heat at 95C
95C
5’ 3’
3’ 5’
55C 3’ 5’
Step 2: Anneal
Primers bind at lower temp 55C
5’ 3’
3’ 5’ 5’ 3’
72C
Step 3: Extend
Taq polymerase extends primer 3’OH
at 72C (dNTPs and Mg++)
Step 4: Repeated rounds of D, A, E
5’ 3’
3’ 5’

47. PCR Number of Target Molecules Created1 2 3 4 5 8 6 16 7 32 64 9
128 10
256 11
512 12
1024 13
2048 14
4096 15
8192
16,384 17
32,768 18
65,536 19
131,072 20
262,144 21
524,288 22
1,048,576 23
2,097,152 24
4,194,304 25
8,388,608 26
16,777,216 27
33,544,432 28
67,108,864 29
134,217,728 30
268,435,456 31
536,870,912
1,073,741,824
Cycle Number
Number of Double-stranded Target Molecules
Bank account paying 100% interest every 5 minutes
Swimming pool - 10 drops

48. Relative power of tests
Test type time power
RFLP-VNTR weeks +++ *
PCR:
DQAlpha- macroarray 1 day +
PM - macroarray 1 day ++
D1S80 - gel- VNTR 2 days ++
STRs -gel,CE, arrays 2 days +++
mtDNA- gel, CE, arrays 2 days +
alu -gel, CE, arrays 2 days ++
* not useful on degraded DNA

49. DNA Chant Review
The subject of our class today
Is simply stated DNA
Sugar-Phosphate backbone chains
Hold the base pairs here’s their names
Chorus: AT- AT
GC- GC
ATGC, ATGC (together)
RFLP holy graile
Put bad guys away in jail
PCR can lend a hand
Amplifying those weak bands >Chorus
Blood, saliva, semen too,
Can be used as crucial clues
Fingernails and skin and hair
DNA is everywhere >Chorus

50. Summary 1 Why study DNA DNA Biology and Genetics
Law enforcement, evolution, agricultural, and human applications-medical diagnostics
DNA Biology and Genetics
DNA is contained in cells –the basic unit of life
Found in nuclei, mitochondria and chloroplasts
Organized in chromosomes. Located at positions called loci and come in different forms or alleles.
Homozygous if the same, heterozygous if different
Alleles segregate independently and assort randomly when on different chromosomes. Random assortment is desired for forensic DNA loci.
DNA Function and Structure
DeoxyriboNucleic Acid : blueprints of life
Replication, Information storage and mutation RIM
Central Dogma
DNA >RNA------>protein
transcription translation

51. Summary 2 DNA Structure and Function continued: DNA Replication
Bases are Adenine, Guanine, Cytosine and Thymine- Asian Guys Can Teach: AGCT
Base pairing is A to T and G to C- DNA is where its AT
Sequence of Bases Store information- Like the sequence of numbers in a Phone Number
Sides of the ladder are Sugar-Phosphate backbones
Nucleotides are the building blocks (dNTPs) themselves made of phosphate base and sugar= PBS- The only station Sierra and Gabriel can watch
DNA base pairs- DNA velcro (David Letterman
DNA Replication
Semi-conservative- Half republican (old) /half democrat (new)
Template directed with base pairing (AT, GC)
5 required ingredients of PCR - primer, template, Mg, dntps, DNA polymerase (PTMDD)

52. Summary 3
Steps in forensic DNA typing are – Evaluation, Extraction, Quantification, Typing and Interpretation
1) Evaluation- Is it there? Screen- blood? Semen? Saliva, human?
2) Extraction- Get and clean DNA
Open cells -Get DNA
Organic, Chelex and FTA extractions
Quantify- Determine quality and quantity?
How good and how much did you get?
Yield Gels, Slot Blots, Real time PCR assays