dna finger printing- khz
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DNA Fingerprinting and Forensic
Analysis
Khizar Hayat Bhatti
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What is a DNA fingerprint?
Every cell of an individual carries a copy of
the DNA
a cell collected from a persons skin or hair
folicle contains the same DNA as from that
persons heart tissue or white blood cells
Order of base pairs in the DNA of every
individual is different except identical
twins
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Forensic DNA?
Forensic science is the application of scienceto law
previous technologies used
photography, video cameras, A/Vtapes
fingerprinting
new technologies
DNA fingerprints
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Overview: DNA tests can be for:
A. Prosecution
B. Defense
C. Post-conviction testing
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DNA Technology in Court
Criminal ProsecutionUnprecedented sensitivity and
specificity for typing biological
samples
Growing use of databanks and
dragnets to identify suspects
Rapidly becoming cheaper and
faster
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Sources of Biological Evidence
Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Tissue
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Possible DNA Sources
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Types of objects where DNA may be
found
Blood Stains
Semen Stains
Chewing Gum
Stamps & Envelopes
Penile Swabs
Plant Material
Sweaty Clothing
Bone
Hair
Fingernail Scraping
Saliva
Animal Material
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Brief History of Forensic DNA Typing
1980 - Ray White describes first polymorphicRFLP marker
1985 - Alec Jeffreys discovers multilocusVNTR probes
1985 - first paper on PCR 1988 - FBI starts DNA casework
1991 - first STR paper
1995 - FSS starts UK DNA database 1996First mtDNA case
1998 - FBI launches CODIS database
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DNA Use in Forensic Cases
Most are rape cases or murders
Looking for match between evidence
and suspect
Must compare victims DNA profile
Mixtures must be resolved
DNA is often degradedInhibitors to PCR are often present
Challenges
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Human Identity Testing
Forensic cases -- matching suspect with evidence
Paternity testing -- identifying father
Historical investigations-Czar Nicholas, Jesse
James Missing persons investigations
Mass disasters -- putting pieces back together
Military DNA dog tag Convicted felon DNA databases
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Short Tandem Repeats (STRs)
the repeat region is variable between samples while the
f lanking regions where PCR pr imers bind are constant
7 repeats
8 repeats
AATG
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another
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Short Tandem Repeat
AGAT AGATAGAT
AGAT
AGAT
AGAT
AGAT
AGAT
AGAT
AGAT
6
4
DNA Profile =4,6
TCTA TCTATCTA
TCTA
TCTA
TCTA
TCTA
TCTA
TCTA
TCTA
7
5
DNA Profile =5,7
TCTA
TCTA
STR
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Multiplex PCR
Over 10 Markers Can Be
Copied at Once
Sensitivities to levels less than
1 ng of DNA
Ability to Handle Mixtures
and Degraded Samples
Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping Size
Ranges
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An Example Forensic STR Multiplex Kit
D3 FGAvWA 5-FAM (blue)
D13D5 D7 NED (yellow)
A D8 D21 D18 JOE (green)
GS500-internal lane standard
ROX (red)
AmpFlSTRProfiler PlusKit available from PE Biosystems (Foster City, CA)
9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction
100 bp 400 bp300 bp200 bp
Size Separation
Colo
rSeparation
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Short Tandem Repeats (STRs)
the repeat region is variable between samples while the
f lanking regions where PCR pr imers bind are constant
AATG
7 repeats
8 repeats
AATG AATG
Primer positions define PCR product size
Fluorescentdye label
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Short Tandem Repeats (STRs)
2. Gel-based systems
with Fluorescent
Detection
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DNA Quantization
using Slot Blot
AMEL
D3
TH01TPOX
Penta D
Penta EFGAD21 D18
CSF
D16D7
D13D5
VWA D8
PCR Amplification with Fluorescent STR Kits and
Separation with Capillary Electrophoresis
Blood Stain
verview of Steps Involved in DNA Typing
Genotyping by Comparison to Allelic Ladder
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ABI Prism 310 Genetic Analyzer
capillary
Syringe withpolymer solution
Autosampler
trayOutlet
buffer
Injection
electrode
Inlet
buffer
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Chemistry Involved
Injection
electrokinetic injection processimportance of sample preparation (formamide)
Separation
capillaryPOP-4 polymer
buffer
Detection
fluorescent dyes with excitation and emissiontraits
virtual filters (hardware/software issues)
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DNA Technology in Court
Criminal Defense
Unprecedentedsensitivity and
specificity fortyping biologicalsamples
Potential support
for alternativetheories of the case
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DNA Technology in Court
Post-conviction exonerations (2008 in US)based on DNA evidence have revealed
problems with the justice system
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Sources of Error
Saks & Koehler,
Science(2005)
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Mid-1980s: The Colin Pitchfork Case
Two young women were raped and
murdered in Narborough, England
5,000 local men are asked toprovide blood/saliva samples
1st exoneration and conviction
on forensic DNA evidence
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Three generations of DNA testing
DQ-alpha
TEST STRIP
Allele = BLUE DOT
RFLP
AUTORAD
Allele = BAND
Automated STR
ELECTROPHEROGRAM
Allele = PEAK
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Two relatively new DNA tests
Mitochondrial DNA
mtDNA sequence
Sensitive but not
discriminating
Y-STRs
Useful with mixturesPaternally inherited
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DNA content of biological samples:
Type of sample Amount of DNA
Blood 30,000 ng/mL
stain 1 cm in area 200 ng
stain 1 mm in area 2 ng
Semen 250,000 ng/mLPostcoital vaginal swab 0 - 3,000 ng
Hairplucked
shed
1 - 750 ng/hair
1 - 12 ng/hair
Saliva
Urine
5,000 ng/mL
1 - 20 ng/mL
2
2
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Crime Scene Samples &
Reference Samples
Differential extraction in sex
assault cases separates out DNA
from sperm cells
Extract and purify DNA
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Differential Extraction of Semen Stain
Female Extract Male Extract
Graphic from Inman & Rudin, An Introduction fo Forensic DNA Analysis. CRC Press.
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Blood
Hair Roots
Saliva
SweatTissue
Chemical
DNA
Isolation of DNA
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PCR Amplification
Groups of amplified STR products are labeled
with different colored dyes
(blue, green, yellow)
DNA regions flanked by
primers are amplified
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The ABI 310 Genetic Analyzer
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ABI 310 Genetic Analyzer: Capillary Electrophoresis
Amplified STR DNA injectedonto column
Electric current applied
DNA separated out by size:
Large STRs travel slower
Small STRs travel faster
DNA pulled towards the
positive electrode
Color of STR detected andrecorded as it passes thedetector
Detector
Window
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Why the Y Chromosome? Applications
forensic investigations (98% of violent crime by men) genealogical purposes
evolutionary studies
Advantages to Human Identity Testing
male component isolated without differential extraction
paternal lineages
Needs
population studies to evaluate diversity of haplotypes
robust assay for accurate characterization of Y markers
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Y-STRs
Problem:
~99% of violent crimes are committed by
men
DNA Mixtures of male suspect and female
victim can pose an analytical challenge,
especially when the female contribution ismuch greater than the male = preferential
amplification
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Y STR Multiplex Assay
100 bp 400 bp300 bp200 bp
DYS19 389II389I
390Primer Amounts Dye
Y19 0.25 M JOE
Y389 0.125 M FAM
Y390 0.25 M JOE
Prinz et al. 1997
(Forensic Sci Int, vol. 85, pp.
209-218)
Quadruplex I
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FBI E l ti f M fi ld E
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FBIs Explanation of Mayfield Error
Confirmation Bias
[B]ecause the initial examiner was a highlyrespected supervisor with many years ofexperience, it was concluded that
subsequent examinations were incompleteand inaccurate. To disagree was not anexpected response. Robert B. Stacey, A report on the erroneous fingerprint
individualization in the Madrid Train Bombing Case. 54J.Forensic Identification 706 (2004).
See, Thompson & Cole, Lessons from the Brandon MayfieldCase. The Champion, April 2005
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The Future of Forensic DNA
CODIS
SNPs & Chips
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FBIs CODISDNA Database
Combined DNA Index System
Used for linking serial crimes and unsolved
cases with repeat offenders
Launched October 1998 Links all 50 states
Requires >4 RFLP markers
and/or 13 core STR markers Current backlog of >600,000 samples
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The Mystical Power of CoDIS
Extremely powerful investigative
tool, linking crimes, and pulling
suspects out of thin air!
Can prevent, as well as solve
crimes!
13 CODIS Core STR Loci
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13 CODIS Core STR Loci
with Chromosomal Positions
CSF1P
O
D5S81
8
D21S1
1
TH0
1
TPOX
D13S3
17
D7S82
0
D16S5
39
D18S5
1
D8S11
79
D3S13
58
FGA
VW
A
AMEL
AM
EL
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STR Analysis by Hybridization on Microchips
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STR Analysis by Hybridization on Microchips
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How to distinguish one persons DNA from another?
We do not need to sequence the entire 3 billion bp Intron regions of DNA (junk DNA) contain
sequences that are 20-100 bp in length, repeated at
different locations (loci) along the chromosome. CGGCTACGGCTACGGCTA (repeated 3 times
at this location; at another location, it may be
repeated 9 times)
These sequences are called Short Tandem Repeats
(STRs) or VNTRs(Variable No. of Tandem
Repeats
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The uniqueness of an individuals STRs
provides the scientific marker of identity
known as a DNA fingerprint.
In the United States the FBI has standardized a
set of 13 STR assays (13 different locations on
the chromosomes) for DNA typing, and hasorganized the CODIS database for forensic
identification in criminal cases.
The United States maintains the largest DNAdatabase in the world: The Combined DNA Index
System, with over 60 million records as of 2007.
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Preparation of a DNA fingerprint
Step 1
Specimen collection
blood, semen, etc
easy to contaminate a DNA sample with DNA fromother sources (bacteria, DNA of person collecting
sample)
DNA is not stable for very long-it degrades
sunlight
heat
moisture
DNA fingerprinting is a comparative
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DNA fingerprinting is acomparative
process:
DNA from crime scene is compared with DNA
of a suspect
So minimum of two samples must be prepared
Step 2
DNA extraction
standardized methods have been developedneed to separate DNA from other cell material
and debris from crime scene.
St 3
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Step 3
PCR using primers targeting STRs at
different loci
PCR amplify STRs using target sites on
chromosome
tep
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tepPCR amplification of DNA
1 strandof DNA
Heat todenature
double-
stranded
DNA
Design primers that anneal to STR locus
Amplify all the regions of the chromosome
where the STRs exist.
STR locus
STR locus
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PCR allows you to make
millions of copies of the STR
region from a single copy of
DNA you recovered from crimescene.
Restriction Fragment Length Polymorphism
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Restriction Fragment Length Polymorphism
G-G-C-C-X-X-X-G-G-C-C-X-X.. G-G-G-C-C-X-X-G-G-C-C-X-X..
STR
C-C-X-X-X-G-GC-C-X-X-G-G
PCR amplifySTR region
STR
well well
Gel
electrophoresis
Person A Forensic sample
For 1 STR sequence at 1 locus
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If you do this for 13 different repeat sequences
at 13 different loci on the chromosome, each
person produces a different band pattern when
the fragments are separated by gel
electrophoresis
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Different
STRs at
other loci
STR1
STR2
STR3
Do any of the
individuals
compare with
forensic sample?
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Dot Blot
C-G-T-Abiotin
G-C-A-T.
Probe made
from sequenceobtained from
forensic sample
Single strand of HLA gene amplified DNA from sample
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Binding of probe to
complementary DNA
G-C-A-T.C-G-T-A
biotinBindingtakes place
C-G-T-Cbiotin
Probe 3No binding
takes place
Wash a a nreacted probe
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Wash away unreacted probe
and add biotin-reactive enzyme
G-C-A-T.C-G-T-A
biotin
Strepavidin
(colorless
enzyme)
Colorless substrate
Colored product (spot lights up)
Dot Blot
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Dot Blot
A visual signal is
produced whenthe different
probes anneal
(bind) to thecomplementary
sequence in the
DNA sample
C-G-T-Abiotin
Probe 1
C-G-T-Tbiotin
Probe 2
C-G-T-Cbiotin
Probe 3
Crime scene
PCR amplified
DNA on each
spot
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What do we end up with?
Blot strips show apattern of spots that
either light up or
remain dark Compare pattern
produced from crime
scene DNA to patternproduced from suspect
DNA
Scene DNA Suspect DNA
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DNA profile database
CODIS Combined DNA Index System
run by FBI
contains profiles of convicted offenders
contains unidentified DNA taken from crime
scenes
visit CODIS website to see how it works
www.fbi.gov/hq/lab/codis/index1.htmCODIS allows identifying possible suspects
when no prior suspect exists
I i f i
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Invasion of privacy
Some groups are worried that DNA samples will
get in hands of insurance companies or potentialemployers
use to identify genetic defects that might cost them $$
Why is this concern invalid?What do you need to identify a genetic defect?
What does the STR analysis yield in the way of datathat can provide information on genetic disorders?
Some groups are demanding that DNA samplesbe destroyed after investigation is complete.
Is this a good idea?
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Meeting Legal Standards
Court uses 5 different standards to
determine whether evidence should be
allowed in court New technique must meet one or several of
the standards before evidence using new
technique can be introduced.
5 St d d
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5 Standards Relevancytest
Frye standard-general acceptance test
Coppolino standard-allows new or controversial science
to be used if adequate foundation can be laid. Expert
witnesses used in this case.
Marx standard-court must be able to understand and
evaluate scientific evidence. A university professor
may be brought in to give a lecture of the concept.
Daubert standardrequires special pretrial hearings forscientific evidence. Scientific procedure must be
described in a peer-reviewed journal
Simpson/Goldman Murder
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Simpson/Goldman Murder
Pretrial hearings announced that blood collected
at crime scene matched that of O.J.s
Defense argued that contamination could have
occurred during sample collection and between
collection of different samples Technician admitted mislabeling samples
Possibility that evidence might be tainted was
obvious to both the court and the jury
DNA evidence was not allowed as evidence
When rules of evidence are not followed, DNA
sam les lose their value in court.
Chain of custody
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Chain of custody
Requires that collection of evidence must be
systematically recorded and access to evidencemust be controlled
Special challenges for DNA samples
crime scene may have DNA from people other thanperpetrators of crime. These people could become
suspects based on this DNA
DNA collected from victims in a morgue can become
contaminated by DNA of other bodies previously onautopsy table
during early days all procedures for processing DNA was
not standardized, people running assays were not
experienced and made mistakes
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Common Technical Problems
Band shifting
Add DNA
samples from
crime scene and
suspects
+
-
Gel is not of uniform porosity
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Maintaining High Standards
American Society of Crime LaboratoriesDirectors
National Forensic Science Technology
Center College of American Pathologists
Forensic Laboratory at CEMB-LHR
All provide accreditation to forensic laboratories
Proficiency testing of technicians
Blind tests
Educating the Jury
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Educating the Jury
Comparison of STR data is a statistically-based
methodJurors may not understand significance of a 1 in 50
billion chance of a random match
Attorneys must compare chance of random match ofDNA data with chance that people will die by being
hit by lightening over their lifetime to make them
appreciate these numbers
Jurors must understand what DNA evidenceoffers in the way of putting suspect at a crime
scene
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Paternity testing
Verifying parents of a child to determineresponsibility for child support
250,000 cases per year in U.S.
Using amniocentisis, it is even possible toverify a childs parents before birth
collect fetal cells from amniotic fluid
DNA extraction and fingerprinting.
No longer necessary with PCR technology
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PCR amplification,
then DNA fingerprinting
M th STR
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Offspring STRs
Mothers STRs
STRs of suspected Father
Is the suspect the father?
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M DNA i d i f ili
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MtDNA is used to reunite families
separated by corrupt governments
Junta in Argentina arrested pregnant womenand took their newborn infants and gave them
to supporters of the regime without consent of
mother.
AAAS helped reunite 51 children with their
natural mothers after the Junta regime
collapsed.
MtDNA can be used to identify a buriedcorpse that has been buried for many years
if you have living relatives whose DNA you
can compare it to.
MtDNA and evolutionary biology
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MtDNA and evolutionary biology
MtDNA mutates at a relatively constant rate of2-4% every million years.
Allows scientists to trace gene frequency changes
over time. Eve hypothesis allowed scientists to trace a
majority of people now living on Earth to a common
female ancestor from ancient Africa
Followed human migration and dispersal from that
location to other parts of the world.
Jared Diamonds Guns Germs and Steel
Oth li ti f DNA fi i ti
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Other applications of DNA fingerprinting
Distinguishing between the North Americanand Asian strains of herb ginseng.
The different strains putatively have different
therapeutic effectsAsians want NA strain
Americans want Asian strain
DNA RFLP analysis can distinguish between thetwo (used in this case as a means of monitoring
quality control/quality assurance)
DNA id h h th t th j it
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DNA evidence has shown that the majority
of bison herds have some domestic
livestock as ancestors.No outward (phenotypic) evidence that this is
the case, however.
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The science of DNA profiling is
sound.But, not all of DNA profiling is
science.This is especially true in situations
involving: small amounts of starting
material, mixtures, relatives, andanalyst judgment calls.
Careers in DNA testing
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g
Laboratory technicians
must be able to work very meticulously forensic science technicians must pass a test to demonstrate
these skills before being let loose at a crime scene
sometimes have to perform their sample manipulation in a
clean roomRequirements
B.S. degree in biology, biochemistry or molecular biology
or a specialized Associates degree in biotechnology and
laboratory experience. Good writing skills (lab notebook entries)
Good math and communication skills
$
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