JS 113: Introduction to DNA Typing

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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

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Announcements, Assignments and Schedules

• Announcements and Assignments
• Study for quiz
• Extra Credit 1 point each due Monday 19 Nov
• Read Esslinger et al. 2004. Using STR analysis to
  detect human DNA from exploded pipe bomb devices.
  JFS 49(3) http//aafs.micronexx.com/PDF/JOFS/JFS2
  003127/JFS2003127.pdf- I will provide hard copies
  to those interested today.
• http//www.ncjrs.org/nij/DNAbro/intro.html
• 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)

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Top 10 ways you can tell youre a forensic DNA
scientist

• 10. You have your kids 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. 2005 ACS Meeting presentation
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Who Cares?

• 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. 1999 Sci Amer. 28094)
• Understanding the molecular basis of development,
  disease and aging
• Screening candidates for bone marrow/organ
  transplants and grafts

WE ALL DO!
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Report on the DNA Evidence Backlogand 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 casesnot 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
  http//www.fbi.gov/hq/lab/codis/index1.htm

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Presidents DNA Initiative Justice for All act of
2004 www.dna.gov 1 billion dollars in funding
over 5 years From 2004-2009 to address the
backlog and to assist in developing more
discriminating, more cost effective, and faster
methods of DNA typing 98 million just announced
09-19-05
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Butler, J. 2005 ACS Meeting presentation
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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

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Innocence Projecthttp//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.

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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.
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DNA in the CellIn nuclei, mitochondria and
chloroplasts (plants) organized in chromosomes
(wound around histones)DNA double bagging
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I. Intro to DNA Facts and Jargon   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
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DNA function Whats it do?
DeoxyriboNucleic Acid blueprints of
life Replication, Information Storage and
Mutation
Central Dogma information
flow---------------gt DNA-------gtRNA------gtprotei
n transcription translation
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DNA Organization and InheritanceHuman Genome
Contains 23 Pairs of ChromosomesIt is inherited
from your mom and dad
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Basic Chromosome Structureand Nomenclature
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 SSingle copy sequence 459 459th locus
described on C12
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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

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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
Punnett Square
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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?
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Review- Mendelian GeneticsLaw 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.

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• 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
• P P
• S-A T-S
• P P
• S-G C-S
• P
•  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

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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!

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DNA StructureNucleotides 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
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Basic Components of Nucleic Acids
5end Phosphate SugarBase Phosp
hate SugarBase 3end
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DNA Structure2 Complimentary, Antiparallel
Strands held together by Base Pairs- H Bonds
.
AT held with 2 H Bonds
GC held with 3 H Bonds
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Wheres Daddy?
PCR product size (bp)
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Father
12
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Child 1
8
14
Child 2
11
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Child 3
8
12
Mother
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DNA St. Patricks Day Salute to the Molecule of
HeredityFrom Biology 110- UNC 1993 Steve Lee

• The molecular structure today
• Is hereditys 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
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Summary 1

• Why study DNA
• 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-------gtRNA------gtprotein
• transcription translation

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Summary 2

• DNA Structure and Function continued
• 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)

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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

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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
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Review DNA is organized inside the cell nucleus
and mitochondria
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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)
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Differential Extraction MethodFor 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
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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!

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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
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Slot Blot quantification DNA-DNA
HybridizationDNA is where its 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

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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

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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

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DNA Methods

• 1) Extract
• 2) Quantitate
• 3) Distinguish
• Size
• Content
• Restriction Fragment Length Polymorphisms (RFLP)
• Polymerase Chain Reaction (PCR)

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• 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 exceptionsidentical twinsdiseased
  individuals, mtDNA (sport analogs)

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RFLP Role Playing

• 1. Students are assigned a base.
• 2. Sequence is provided with a restriction site
  5GGCC3 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

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PCR based systems are rapid, require less
material than RFLP and less time for typing
Polymerase Chain Reaction PCR is simply
repeated rounds of DNA replication

• Molecular xeroxing
• Calvin and Hobbes example

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5-P

• 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 3OH
  to attach incoming nucleotide

Primer
3-OH
3-OH
dNTP
Template
Mg
DNA polymerase
5-P
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PCR is simply repeated rounds of DNA replication
Step 1 Denature Separate H bonds with heat at 95C
95C
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 3OH
at 72C (dNTPs and Mg) Step 4 Repeated
28-30 rounds of D, A, E
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PCR Number of Target Molecules Created

• Bank account paying 100 interest every 5 minutes
• Swimming pool - 10 drops

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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

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• DNA Chant Review
• The subject of our class today
• Is simply stated DNA
• Sugar-Phosphate backbone chains
• Hold the base pairs heres 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 ----------------gtChoru
  s
• Blood, saliva, semen too,
• Can be used as crucial clues
• Fingernails and skin and hair
• DNA is everywhere ---------------gtChorus

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Summary 1

• Why study DNA
• 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-------gtRNA------gtprotein
• transcription translation

51
Summary 2

• DNA Structure and Function continued
• 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