DNA Finger Printing- KHZ

8/11/2019 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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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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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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Criminal Defense

Unprecedentedsensitivity and

specificity fortyping biologicalsamples

Potential support

for alternativetheories of the case


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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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DNA Finger Printing- KHZ

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