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DNA: the indispensable forensic science tool. What is DNA. Deoxyribonucleic Acid Genetic Code of Life Codes for production of proteins that determines our traits. Where is DNA?. Prokaryotic organisms = bacteria DNA is located as one large circular strand (chromosome) within the cell

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what is dna
What is DNA
  • Deoxyribonucleic Acid
    • Genetic Code of Life
      • Codes for production of proteins that determines our traits
where is dna
Where is DNA?
  • Prokaryotic organisms = bacteria
    • DNA is located as one large circular strand (chromosome) within the cell
  • Eukaryotic Organisms = plants, animals, fungi
    • Condensed DNA forms chromosomes and is found within the nucleus of the cell
      • Humans have 46 chromosomes
      • DNA wraps around proteins called histones to condense and form chromosomes
structure of dna
Structure of DNA
  • Structure is a DOUBLE HELIX or “twisted ladder”
    • DNA is a large macromolecule (polymer) made of nucleotide monomers
      • Each nucleotide is composed of a deoxyribose (sugar), a phosphate group, and nitrogen base
structure of dna1
Structure of DNA
    • Sides of the ladder are made of alternating deoxyribose (sugar) and phosphate groups.
  • Nitrogenous Bases—pairs of molecules that form the rungs of the DNA “ladder”
    • Four types of Bases
      • A (adenine)
      • C (cytosine)
      • G (guanine)
      • T (thymine)
    • Base pairing rules
      • A (adenine) always bonds/pairs with T (Thymine)
      • C (cytosine) always bonds with G (Guanine)
dna at work
DNA at Work
  • DNA is the genetic code for producing all the different proteins needed by the body
    • Roles of proteins
      • Enzymes- speed up chemical reaction
      • Cell Transport- movement of materials in and out of the cell
      • Structural- form parts of the body
      • Fight disease ect…
  • Transcription and Translation
    • Genes are segments of DNA that code for a particular trait
      • Genes are transcribed into mRNA
      • mRNA is then translated by ribosome into the amino acid sequence of proteins
dna replication
DNA Replication
  • DNA Replication- Process in which DNA is copied, process is semiconservative
    • Occurs before a new somatic (body) can be created
      • Each cell needs a copy of instructions
    • Occurs before production of gametes or reproductive cells
        • Sperm
        • Egg
polymerase chain reaction
Polymerase Chain Reaction
  • Laboratory process of making copies of DNA
    • Forensic scientists can make billions of copies of small DNA samples in just a few minutes
    • Process mimics DNA Replication
  • Steps
    • DNA Heated to separate two strands
    • Add primer and lower temperature

-primer = short strand of complementary DNA that will target and attach to original separated strands

    • Add free nucleotides and DNA polymerase

-DNA polymerase attaches free nucleotides to remaining original strands to form new “complementary strand

    • result is two molecules of DNA that are identical to original
        • Each has one original strand and 1 new complementary strand, therefore replication is said to be “semiconservative”
restriction enzymes
Restriction Enzymes
  • Chemical Agents that cut DNA at specific sites
  • Many different Restriction Enzymes exist that cut at specific locations
    • Example
      • EcoRi
        • G A A T T C

C T T A A G

rflp s restriction fragment length polymorphisms
RFLP’sRestriction Fragment Length Polymorphisms
  • Takes advantage of the fact RESTRICTIONENZYMES will cut DNA at specific sites
  • Specific RESTRICTION ENZYMES can be used to cut into DNA at these specific locations
  • Creates different sized fragments that can be separated by gel electrophoresis
rflp analysis
RFLP Analysis
  • R – Restriction enzymes are used to cut known segments of DNA into
  • F – Fragments that are many different
  • L – Lengths and exhibit
  • P – Polymorphisms, which is the Greek term meaning many sizes. The length of the fragments will vary greatly among individuals
dna fingerprinting
dna fingerprinting
  • Uses process of gel electrophoresis
    • DNA sample that has been cut is place into wells at ends of gel agarose
    • Gel agarose (jello like substance) used to separate DNA into different sized fragments
    • Creates pattern (fingerprint)
steps of dna fingerprinting
Steps of DNA Fingerprinting
  • Extraction

-Cells are isolated and treated with chemicals to release chromosomes from the nucleus

-Chromosomes are unwrapped and DNA is unraveled

*enzymes destroy histones in which DNA is coiled around

  • DNA is cut with restriction enzymes

-different restriction enzymes recognize and cut at different sequences

example: EcoRi recognition sequence = GAATTC

steps of dna fingerprinting1
Steps of DNA Fingerprinting

3. DNA fragments are separated by the process of gel electrophoresis

-DNA negatively charged and attracted to positive end

-shorter fragments travel farther

4. DNA stains are used to show banding pattern or

-probes (often radioactive) that attach to DNA can be used and a film can later be developed to show pattern

dna fingerprinting1
DNA Fingerprinting
  • DNA fingerprinting can
    • match crime scene DNA with a suspect
    • determine maternity, paternity, or match to another relative
    • eliminate a suspect
    • free a falsely imprisoned individual
    • identify human remains
matching dna samples rflp
Matching DNA SamplesRFLP
  • Matching
    • Two samples that have the same band pattern are from the same person
    • No two people will show identical band patterns, except identical twins
paternity and maternity determination rflp
Paternity and Maternity DeterminationRFLP
  • Inheritance Matching
    • Each band in a child’s DNA fingerprint must be present in at least one parent
rflp limitations
RFLP Limitations
  • Although DNA fingerprinting and the use of RFLP’s is very useful it no longer the preferred method for DNA profiling.
    • Must have a complete DNA sample
    • Hard to copy complete DNA samples using PCR
    • STR analysis is now the preferred
genes and chromosomes
Genes and Chromosomes
  • Genes are functional units or segments of DNA that code for proteins
  • Genes are located on chomosomes
  • Humans have 46 chromosomes
    • Inherit 23 from each parent
      • 22 regular chromosomes (autosomes)
      • 1 sex chromosome (X or Y)
junk dna
Junk DNA
  • The HUMAN GENOME contains non-coding regions called junk DNA
    • Junk DNA can be found between functional genes
    • Junk DNA even be found within our genes
      • many randomly repeated sequences (VNTR’s and STR’s) are located within genes or coding , functional DNA sites(loci)
      • number of randomly repeated sequences varies between individuals at each site
vntr variable number of tandem repeats
VNTRVariable Number of Tandem Repeats
    • Short segments of randomly repeated sequences of junk DNA
  • Located within genes or coding DNA at specific sites or loci
  • Usually between 9-80 base pairs long
  • Number of repeats at specific sites varies from person to person
str s short tandem repeats
STR’sShort Tandem Repeats
  • Even shorter segments of randomly repeated
  • Short repeat themselves
      • Usually 3-7 base pairs long
        • Example: GATA
    • Number of times it repeats itself in a sequence varies from person to person (like VNTR’s)
dna profiling using str s
DNA Profiling Using STR’s
  • STR’s within a gene locus (location) are counted
    • Number of short tandem repeats at each site varies from person to person
  • 13 different gene sites or loci are used in analysis
dna profiling str s
DNA Profiling STR’s
  • PCR is used to amplify or make copies of the DNA sample
  • Genes or Loci are isolated and then cut with a restriction enzyme
  • Segments are separated to determine how many times the STR occurred within the gene locus
    • Each person has two gene sites for each of the 13 loci
      • Inherit one from each parent
    • Number of repeats at each site is analyzed and compared
      • Example 12/8, 10/8, or 6/ 4
    • Preferred Current Method Used In PROFILING
    • Easier to extract, cut, and copy amplify from DNA of individual using PCR
  • Can profile even if there is Deteriated or incomplete DNA sample available
13 codis str s probability of 2 people matching
13 CODIS STR’s Probability of 2 People Matching

STR African American Caucasian

1 D3S1358 .094 .075

2 vWA .063 .062

3 FGA .033 .0364 TH01 .109 .081

5 TPOX .090 .1956 CSF1PO .081 .112

7 D5S818 .112 .158

8 D13S317 .136 .085

9 D7S820 .080 .065

10 D8S1179 .082 .067

11 D21S11 .034 .039

12 D18S51 .029 .028

13 D16S539 .070 .089

probability and str analysis
Probability and STR Analysis
  • What are the chances of two Caucasian individuals being matches for the STR vWA
  • What are the chances that two African American individuals are matches for the STR’s FGA and TP0X?
  • What are the chances of two Caucasian individuals being matches for the STR’s vWA, TH01, and, D18S51?
  • What are the chances of two African American individuals being matches for the STR’s TH01, FGA, vWA, and D18S51?
str analysis
STR Analysis
  • So how unique is our DNA? The chances of the number of STR’s being exactly the same at each of the 13 sites is…
      • 1/575 trillion for Caucasian Americans
      • 1/900 trillion for African Americans
sex identification
Sex Identification
  • Focus is on the amelogenin gene which is located on the X and Y chromosome
    • Gene codes for tooth pulp
    • Gene on Y chromosome is 6 bases shorter on X chromosome than the on the Y
      • Females, XX, show only one band pattern when gene is amplified (copied through PCR) and separated through electrophoresis
      • Males will show 2 band patterns when same process is completed
codis
CODIS
  • Combined DNA Index System
    • 13 STR’s are used for profiling and determining matches
      • Computer Program Contains Database of DNA profiles
      • States are mandated to report …
        • DNA Profiles of convicted sex offenders and other criminals (convicted of certain crimes)
        • DNA Profiles of unsolved cases
        • DNA of missing persons
    • Law Enforcement Agencies can run DNA from new crime scenes through CODIS to determine possible matches
mitochondrial dna
Mitochondrial DNA
  • Mitochondria are cell structures found in all humans
  • Mitochondria are the “powerhouses” of the cell and contain they contain DNA
    • This is separate from the nuclear DNA of the cell and ONLY contains DNA from ONE PARENT.
    • mDNA is inherited through the ova (egg) from mother
  • This mDNA of MtDNA is inherited maternally
    • From the mother
    • Not as exclusive, DNA is the same as all those maternally related.
      • Siblings, cousins, all persons ect… share common DNA if maternally related.
  • mDNA can often be found in samples or remains that do not contain nuclear DNA
    • Hair shaft, bones, ect…
collection of biological evidence
Collection of Biological Evidence
  • What can DNA be obtained from?
    • Blood
    • Semen
    • Epithelial (Skin) Cells from direct transfer
    • Sweat (epithelial cells)
    • Saliva (epithelial cells)
  • Latex Gloves must be used to collect specimens thought to contain DNA
    • Separate gloves for each piece of evidence
      • Prevents contamination
packaging preservation of dna evidence
Packaging & Preservation of DNA Evidence
  • Samples must be allowed to dry and/or placed in breathable container
    • Avoids breakdown of DNA by bacteria
  • A small portion of the sample must be collected and packaged separately, known as substrate control
    • used for comparative purposes later
      • If does not match then DNA sample is invalid
collection of dna from suspects
Collection of DNA from Suspects
  • Buccal Swap
    • Cotton swab used to rub inside of cheek
      • Transfer of epithelial cells to tranfer
  • Specimens must be collected and preserved in same manner previously discussed
    • Separate gloves each sample
    • Substrate control
dna backlog
DNA Backlog
  • # of cases involving DNA evidence far exceeds the processing abilities of crime labs
  • Many crime labs are backlogged several months
  • Other pieces of evidence, blood ect… are often used first to determine whether to include or exclude a suspect
    • If other evidence includes suspect then DNA evidence is processed to add INDIVIDUALIZED evidence to the case