Download DNA: the indispensable forensic science tool

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
• 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 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 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 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- 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
• 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
1
2
3
•
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
• Chemical Agents that cut DNA at specific sites
• Many different Restriction Enzymes exist that
cut at specific locations
– Example
• EcoRi
– G A ATT C
C T TA AG
RFLP’s
Restriction Fragment Length Polymorphisms
• Takes advantage of the fact RESTRICTION
ENZYMES 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
• 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
• 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
1. 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
2. DNA is cut with restriction enzymes
-different restriction enzymes recognize and cut
at different sequences
example: EcoRi recognition sequence = GAATTC
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
Sample DNA Fingerprint Analysis
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
– 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
• Inheritance Matching
– Each band in a child’s DNA fingerprint must be
present in at least one parent
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 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
• 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
• 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
• 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
• 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
• 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
1
2
3
4
5
6
7
8
9
10
11
12
13
STR
D3S1358
vWA
FGA
TH01
TPOX
CSF1PO
D5S818
D13S317
D7S820
D8S1179
D21S11
D18S51
D16S539
African American
.094
.063
.033
.109
.090
.081
.112
.136
.080
.082
.034
.029
.070
Caucasian
.075
.062
.036
.081
.195
.112
.158
.085
.065
.067
.039
.028
.089
Probability and STR Analysis
1) What are the chances of two Caucasian
individuals being matches for the STR vWA
2) What are the chances that two African American
individuals are matches for the STR’s FGA and
TP0X?
3) What are the chances of two Caucasian
individuals being matches for the STR’s vWA,
TH01, and, D18S51?
4) What are the chances of two African American
individuals being matches for the STR’s TH01,
FGA, vWA, and D18S51?
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
• 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
• 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
• 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
• 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
• 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
• 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
• # 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