DNA DeoxyRibonucleic acid

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DNA

• Three billion base pairs in DNA
• Human cells-23 pairs of chromosomes except
  gametes and rbcs
• 99.5 similarity among individuals
• Genome-total DNA in the cell
• 5 of genome is encoded-directs synthesis of
  proteins
• 95 is non-encoded-doesnt directly code for
  protein-Junk DNA

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Polymorphisms

• Vary in length (length polymorphism) and base
  sequence
• Two sequences
• 1. Variable Number Tandem Repeats (VNTRs)-same
  base sequence repeats throughout a specific
  locus-can be hundreds of base pairs
• 2. Short Tandem Repeats (STRs)-3-7 bp long-repeat
  throughout loci-ex.
• AGTT AGTT AGTT AGTT AGTT AGTT

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Techniques of DNATyping

• 1. Restriction Fragment Length Polymorphism
  (RFLP)-used restriction enzymes to cut DNA into
  fragments-individuals differ in where these
  enzymes cut-different fragment sizes based on
  VNTRs
• 2. Polymerase Chain Reaction (PCR)-STR containing
  regions amplified

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DNA Fingerprinting

• Alec Jeffreys-1985-areas of DNA exhibit
  polymorphism using RFLPs
• These polymorphic areas allow scientists to
  identify individuals
• DNA Fingerprinting-process of isolating and
  analyzing the DNA

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DNA RFLP typing process
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Intertwined strands of DNA representing segments
of two chromosomes Chromosome segment on the
left contains three repeating sequences of
TAG Chromosome segment on right has two
repeating sequences of TAG
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 Variants of STR TH01 Top DNA strand-six repeats
of sequence AATG Bottom DNA strand-eight
repeats of sequence AATG
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• http//wps.prenhall.com/wps/media/objects/3801/389
  2550/DNACD_mod07-2-08.swf
• http//wps.prenhall.com/wps/media/objects/3801/389
  2550/DNACD_mod02-1-12.swf
• http//wps.prenhall.com/wps/media/objects/3801/389
  2550/DNACD_mod02-1-13.swf

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Process of DNA Typing

• DNA Extraction
• DNA Purification
• DNA Quantitation
• DNA Amplification-PCR
• Fragment Analysis-Capillary Electrophoresis
• Evaluation-Electropherogram

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DNA Extraction and Purification

• Extraction of DNA Sample-remove DNA from
  cells-DNA Extraction Machine
• Purification-Separate DNA from contaminants

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Portable Genetic Analyzer
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Quantitation

• Quantitation-Determine amount of DNA in the
  sample
• QuantiBlot quantitation-determines amount of DNA
  extracted from original sample-hybridization
  probes and colorimetric or chemiluminescent
  detection.

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RT-PCR

• RT PCR amplifies DNA sample and studied during
  real time, or while it happens.
• Data is collected during the process as it
  analyzes changes of amount of fluorescence from
  DNA amplification
• TaqMan Probes-labeled with two fluorescent dyes
  sensitive to different wavelengths-hybridize to
  DNA that is amplified.-probe checks for changes
  in fluorescence caused by displacement of
  double-dyed probe from a specific area in
  amplified DNA.
• Quantitation of DNA and determines condition of
  DNA present
• Amplifies that DNA.
• Real Time PCR is more sensitive than
  Quantiblot-risks of contamination are higher
• The Quantiblot system-10 to 0.15 ng of DNA
• RT PCR -from 0.003 to more than 50 ng/microliter
  of DNA
• 1 nanogram one billionth of a gram

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DNA Amplification-PCR

• Polymerase chain reaction (PCR)-make copies of
  loci with STRs-makes enough DNA to be detected
• The DNA sample is denatured, cooled, primers
  annealed, free nucleotides synthesize
  complementary strands of DNA
• This process is repeated for about 28 cycles
• Taq DNA polymerase-attaches nucleotides to DNA
• DNA primers initiate replication
• PCR rxns include template DNA which will be
  amplified and deoxynucleotide triphosphates that
  will form the new DNA strands

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PCR
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Thermalcycler-A PCR Machine The PCR Song
http//bio-rad.cnpg.com/lsca/videos/ScientistsForB
etterPCR/
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Automated Process of PCR

• a. Heated to 95o C- Denature (separate) DNA into
  two strands
• b. DNA primers hybridize to complementary bases
• c. DNA Polymerase binds to primer and adds bases
  of DNA
• Process repeats-one cycle-4 strands,2-8
• http//www.sumanasinc.com/webcontent/animations/co
  ntent/pcr.html

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Appropriate primers flanking the repeat units of
a DNA segment must be selected and put in place
in order to initiate the PCR process.
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PCR Animations

• http//www.dnalc.org/ddnalc/resources/shockwave/pc
  ranwhole.html
• http//www.dnalc.org/ddnalc/resources/animations.h
  tml

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Genetic Analyzer

• Fluorescent Detection
• Charge-coupled device-CCD camera detector
  monitors 525-625 nm wavelengths
• Photons fall on pixels-silicon of CCD absorbs
  energy and converts light into electronic charge
• Number of electrons collected-depends on light
  level, exposure, and wavelength
• Separates STR regions by size-laser measures
  fluorescent emissions

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Capillary Electrophoresis

• Fragment Analysis- Electrophoresis- electrical
  current is passed through polymer -DNA moves
  toward positive end and separates into fragments
  by size - Acrylamide polymer (capillary)
  electrophoresis
• Sixteen capillaries used simultaneously- run 16
  different samples in 45 minutes
• STRs lengths differ among individuals
• Smaller STRs move through capillaries faster
• Different loci-different colored
  fluorescence-detected by laser
• Profiles show of repeats at a locus by
  comparison of where fluorescence occurred in
  sample to a standard ladder
• ABI Prism 3130 Genetic Analyzer-uses capillary
  electrophoresis- separates STR segments by size
  and measures them with laser by recording light
  fluorescence
• http//wps.prenhall.com/wps/media/objects/3801/389
  2550/DNACD_mod04-2-08.swf

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Capillary Electrophoresis
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Genetic Analyzer

• Alleles represents as peaks
• Software-Applied Biosystems(ABI) GeneScan and
  GenoTyper
• GeneScan identifies signal corresponding to dyes
  in sample
• Uses GeneScan analysis-identify label peaks
• Electropherogram-Graph representing STRs

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Data Analysis

• Isolate locus of DNA and determine number of STRs
  of given sequence
• Individuals receive different numbers of repeats
  from each parent
• Thirteen loci-probability of two individuals with
  same STRs-one in several hundred trillion

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DNA Analysis

• Data Analysis
• GeneMapperIDTM v3.2 software-Applied
  Biosystems-computer program that automatically
  defines and assigns loci according to a set a
  standard rules
• DNA analyst manually assesses them
• The success of DNA profiling is determined by
  total peak heights, total peak area, and overall
  quality of DNA profiles

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Electropherogram
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Process of DNA Typing

• Evaluation-Computer analyzes data-Electropherogram
  -stored on CD as evidence-compared with suspect
  DNA profile or entered into CODIS
•  

http//wps.prenhall.com/wps/media/objects/3801/389
2550/DNACD_mod04-2-11.2.swf
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http//wps.prenhall.com/wps/media/objects/3801/389
2550/DNACD_mod04-2-11.2.swf
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CODIS

• Combined DNA Index System-database of DNA from
  felons and crime scenes

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Whos your Daddy?Paternity testing

• ABO blood typing-exclude paternity, but cannot
  confirm paternity
• DNA testing can confirm paternity

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Y-STRs

• http//wps.prenhall.com/wps/media/objects/3801/389
  2550/DNACD_mod08-4-01.swf

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The Y Chromosome
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Whos your momma?Mitochondrial DNA

• Passes through maternal lineage
• Hair shaft, teeth, old bones
• Is very stable
• Inherited unchanged from mother
• All cell components of developing zygote come
  from mother
• All cells in body contain identical DNA
• mtDNA mutation-once every 6,500 years
• Used in hair analysis-only living part is
  follicle-can get nuclear DNA there-but if hair is
  cut-can get mtDNA from dead cells

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Every cell -hundreds of mitochondria Each
mitochondrion-many copies of DNA Differences
between individuals in mtDNA-two specific
segments HV1 and HV2
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• DNA Evidence Collection at Crime Scene
  Interactive
• http//media.pearsoncmg.com/ph/chet/chet_saferstei
  n_hsforensics_1/cw_le_dna/beg/burglary/burglary_01
  .htm
• http//media.pearsoncmg.com/ph/chet/chet_saferstei
  n_hsforensics_1/cw_le_dna/adv/burglary/burglary_01
  .htm
• Gel Electrophoresis interactive simulation
• http//learn.genetics.utah.edu/units/biotech/gel/

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STR Profiling Sensitivity

• STR Profiling-can detect 125 picograms
• 1 picogramone-trillionth of a gram
• One cell has 7 picograms of DNA
• 18 DNA bearing cells are needed to get a STR
  profile
• Low Copy Number (LCN) DNA-less than 18 DNA
  bearing cells

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Amounts of DNA

• RFLP/VNTRs- 50 ng-1000 ng 1985-1995
• PCR/STRs- 0.5-2 ng 1991 - present
• LCN/STRs lt0.1 ng 1999-present