dna fingerprinting and gel electrophoresis

8/20/2019 DNA Fingerprinting and Gel Electrophoresis

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DNAFINGERPRINTING

AND GELELECTROPHORESIS

Noynay, Rigil KentGuyha, Chuck !ail"illa!in, #e$e!ie DaleDingla%a, E!icka &inette #u$a$il, K!i%%hia &haeLini%, Shanyne

'!(i)ton*o, #ianne #ah*iel


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- any of several similar techniques

for analysing and comparing DNA- used especially in lawenforcement to identify suspects

from hair, blood, semen, or otherbiological materials found at thescene of a violent crime

DNA +nge!!inting

o! DNA !o+ling


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- it depends on the fact that notwo people, have exactly thesame DNA sequence, and thosesegments will be statistically

unique.Fingerprint

- very unliely that any twopeople would have exactly thesame DNA

information.


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!he test is used to determine

whether a family relationshipexists between two people, toidentify organisms causing adisease, and to solve crimes.


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!he procedure for creating aDNA "ngerprint consists of "rst

obtaining a sample of cells, suchas sin, hair, or blood cells,which contain DNA. !he DNA isextracted from the cells andpuri"ed.


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Poly$o!hi%$


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A polymorphism is a clinicallyharmless DNA variation thatdoes not a$ect the phenotype.

At the molecular level,polymorphism is a variation in

nucleotide sequence from oneindividual to another.


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%olymorphisms often occur inthe intervening sequences thatdo not code for proteins. &Note'

(nly a few percent of the humangenome actually encodesproteins.)

All the common blood types,such as the A*( blood groupsystem , are genetic

polymorphisms.


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Tan*e$ !eeat%


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%olymorphism in chromosomalDNA can arise from thepresence of a variable number

of tandem repeats .!hese are short sequences of

DNA at scattered locations inthe genome, repeated intandem +one after another.


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!he number of these repeat unitsvaries from person to person, but isunique for any given individual and,therefore, serves as a molecular

"ngerprint.leavage by restriction enymes

yields fragments that vary in lengthdepending on how many repeated

segments are contained in thefragment.

/ariation in the number of tandem

repeats can lead to polymorphisms.


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0ingle base changesin DNAAbout 123 of human genome

variation comes in the form of single-

nucleotide polymorphisms, +0N%s,pronounced 4snips5, that is,variations that involve 6ust onebase .

!he alteration of one or morenucleotides at a restriction site canrender the site unrecogniable by aparticular restriction endonuclease.A new restriction site can also be


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7n either case, cleavage with anendonuclease results in fragmentsof lengths di$ering from thenormal, which can be detected byDNA hybridiation.

!he altered restriction site can beeither at the site of a disease-causing mutation or at a site somedistance from the mutation.

8xample ' 0icle cell anaemia.


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Alication o-DNA

Finge!!intin

g

)


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'%e% o- DNA P!o+ling

DNA pro"ling isused to solvecrimes and

medicalproblems


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C!i$eForensic science is the use of

scienti"c nowledge in legalsituations.

!he DNA pro"le of eachindividual is highly speci"c.

!he chances of two peoplehaving exactly the same DNApro"le is 92,222 million to :+except for identical twins.


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iological $ate!ial%

u%e* -o! DNA !o+ling*lood;air

0aliva0emen*ody tissue


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DNA P!o+ling can %ol.ec!i$e%

!he pattern of the DNApro"le is then compared withthose of the victim and the

suspect.


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7f the pro"le matches the suspectit provides strong evidence thatthe suspect was present at the

crime scene +N*'it does not provethey committed the crime.

7f the pro"le doesn=t match the

suspect then that suspect may beeliminated from the enquiry.


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E/a$le

A violent murder occurred.

!he forensics team retrieved ablood sample from the crimescene.

!hey prepared DNA pro"les ofthe blood sample, the victimand a suspect as follows'


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0a% the %u%ect at thec!i$e %cene1

Suspects

Profile

Blood sample from

crime sceneVictims

profile


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Sol.ing &e*icalP!o(le$%DNA pro"les can be used to determine

whether a particular person is the

parent of a child.

A child=s paternity +father andmaternity+mother can be determined.

!his information can be used in %aternity suits

7nheritance cases

7mmigration cases


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E/a$le2 A Pate!nity Te%t

*y comparing the DNA pro"le ofa

mother and her child it is possible

to

identify DNA fragments in thechild

which are absent from the motherand

must therefore have been


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I% thi% $an the -athe! o- thechil*1

Mother Child Man


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Fa$ou% ca%e%

7n >22>8liabeth;urley usedDNA pro"lingto prove that0teve *ing was

the father

of her childDamien


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Fa$ou% Ca%e%

olin %itchfor was the "rstcriminal caught based on DNA"ngerprinting evidence.

;e was arrested in :1?@ forthe rape and murder of twogirls and was sentenced in:1??.


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Fa$ou% Ca%e%

(.. 0impson wascleared of a doublemurder charge in :11B

which relied heavily onDNA evidence.

!his case highlightedlab diCculties.


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S'STAGES OF

DNA

FINGERPRINTING


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:' A cell sample is taen- usually a

chee swab or blood test

>' DNA is extracted from sample

9' leavage of DNA by restrictionenyme- the DNA is broen intosmall fragments

B' 0mall fragments are ampli"ed bythe %olymerase hain eaction-results in many more fragments


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E' DNA fragments are separatedby electrophoresis

@' !he fragments are transferredto an agar plate

' (n the Agar %late speci"c DNA

fragments are bound to aradioactive DNA probe


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?' !he Agar %late is washed free ofexcess probe

1' An x-ray "lm is used to detect aradioactive pattern

:2' !he DNA is compared to otherDNA samples


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0hy i% DNAFinge!!inting i% *one1

DNA "ngerprinting is done to'

Find out who a personGs parents orsiblings are.

!his test also may be used to identify

the parents of babies who wereswitched at birth.

0olve crimes +forensic science

7dentify a body


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Ho3 i% DNA

Finge!!inting i% Done1

DNA "ngerprint - is ind of lie aregular"ngerprint.

Hou are born with it, it is unique to

you +unless you have an identicaltwinI, and you can leave it behindwherever you go.


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*ut unlie a "ngerprint fromyour hand, your DNA

"ngerprint canGt be found by 6ust Jdusting for printsJ liethey do on detective shows.

!o "nd a DNA "ngerprint, a

scientist has to "rst tae theDNA out of the nucleus of acell.


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The cell that i% u%e* to

get a DNA +nge!!int can(e2

a sin cell

a hair root cell

chee cell that gets washed out of

your mouth in your spit.


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!his is because your unique DNAis the same in all of your cells.

!he goal is to analye the DNAin a way that shows scientiststhe tiny di$erences in the DNA

of di$erent people.7n the past, scientists used a

technique called FK%

+estriction Fragment Kength%olymorphism..


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FK% analysis - needs lots of DNA

*ut sometimes only a little is left

behind at a crime scene. 0oscientists found a way to use lessDNA.

!hey wored out a method calledmicrosatellite analysis. *ut scientistswant to "nd ways to use even lessDNA


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!hey also want to "nd a way to speedup the process.

!here are so many samples waiting tobe tested that labs canGt handle themall.

!he wave of the future is something

called Jlab-on-a-chip.JKab-on-a-chip - a credit card sied

machine that you could load a tinysample into on the spot.

- would use tiny tubes andpumps to perform all steps normally done

by hand by scientists.


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A*.antage% o- DNA Finge!!inting

:. 7t is an easy and painless method for the sub6ect beingtested. 7t is less invasive then taing a blood sample

>. 7t is an a$ordable and reliable technique

9. 7t can be conducted in a relatively short amount of time

B. Anyone at any age can be tested with this methodwithout any ma6or concerns

E. !here is a large variety of uses such as in legal claims,missing persons cases, identi"cation for the military, andpaternity and prenatal testing

@. !he technique has used since :1?B, maing it highlydeveloped and improved


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Di%a*.antage% o- DNA Finge!!inting :. !he sample of DNA can easily be ruined during the

process of DNA "ngerprinting, causing the sample tobecome completely useless for testing

>. !he process itself is complex and tedious, and can giveresults that may be hard to interpret

9. !he test needs to be run on multiple samples, anumerous amount of times for ideal accuracy. ommonly,labs run each test twice with four samples.

B. %rivacy issues could occur if the information isnGt eptsecure at the lab. %ersonal information legally can only bereleased with a written order. !his personal information ifleaed, could potentially complicate insurance processes,health care and 6ob prospects for an individual

Alec #e4!ey%


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Alec #e4!ey%

*orn 'Alec ohn

e$reys anuary 1, :1E2

(xford,(xfordshire, 8ngland,Lnited Mingdom

Age ' @E Hearsold


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;e is a professor of genetics at

the Lniversity of Keicester, and hebecame an honorary freeman of the ityof Keicester on >@ November :11>. 7n:11B, he was nighted for services to

genetics.

;e is a *ritish geneticist, who developedtechniques for DNA Fingerprinting andDNA pro"ling which are now usedworldwide in forensic science to assistpolice detective wor and to resolve

paternity and immigration disputes.


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Gel Elect!oho!e%i%

- method for separation andanalysis of macromolecules+DNA, NA and %roteins

and their fragments based ontheir sie and charge.

- used in clinical chemistry to

separate proteins by chargeand


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- method of separation ofnanoparticles

- uses a gel as an anticonvectivemedium and


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DNA Oel electrophoresis - usually performed foranalytical purposes, often after ampli"cation ofDNA via % - may be used as apreparative technique prior to use of othermethods such as mass spectometry ,FK% , % , cloning , DNA 0equencing ,or 0outhern blotting for further characteriation.

8lectrophoresis - a process which enables thesorting of molecules based on

sie

0ieving Q when nuclear acid molecules areseparated by applying an electric "eld to movethe negatively charged molecules through amatrix of agarose or other substances.

%roteins are separated by charge in agarose

Lsing an electric "eld, molecules +such as DNA


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g , + can be made to move through a gel made ofagar or polyacrylamide.

!he electric "eld consists of a negative charge atone end which pushes the molecules through thegel, and a positive charge at the other end thatpulls the molecules through the gel.

!he molecules being sorted are dispensed into awell in the gel material. !he gel is placed in anelectrophoresis chamber, which is thenconnected to a power source.

Rhen the electric current is applied, the largermolecules move more slowly through the gelwhile the smaller molecules move faster. !he

di$erent sied molecules form distinct bands on


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Rhen separating proteins or small nucleicacids +DNA, NA or oligonucleotides the gel isusually composed of di$erent concentrations of

acrylamide and a cross-liner, producing di$erentsied mesh networs of polyacrylamide.

Rhen separating larger nucleic acids +greaterthan a few hundred bases, the preferred matrixis puri"ed agarose.

7n both cases, the gel forms a solid, yet porousmatrix.

Acrylamide - is a neurotoxin and must be handledusing appropriate safety precautions to avoidpoisoning.


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Agarose - composed of long unbranchedchains of uncharged carbohydrate without

cross lins resulting in a gel with largepores allowing for the separation ofmacromolecules and macromolecularcomplexes.

7f charges are not all uniform then,


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7f charges are not all uniform then,the electrical "eld generated by theelectrophoresis procedure will a$ect

the species that have di$erentcharges and therefore will attractthe species according to their

charges being the opposite.

athode - negatively charged

- positively charged specieswill migrate towards it.Anode - positively charged

- negatively charged species

will mi rate towards it.


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Tye% o- Gel in Gel

Elect!oho!e%i%Agarose

- 7t is made from the naturalpolysaccharide polymers extractedfrom seaweed.

- are easily cast and handled comparedto other matrices, because the gelsetting is a physical rather thanchemical change.

0amples are also easily recovered


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0amples are also easily recovered.After the experiment is "nished, theresulting gel can be stored in a plastic

bag in a refrigerator.

- do not have a uniform pore sie, but

are optimal for electrophoresis ofproteins that are larger than >22 Da.

Agarose gel electrophoresis can also be


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Agarose gel electrophoresis can also beused for the separation of DNAfragments ranging from E2 base pair to

several megabases +millions of bases,the largest of which require specialiedapparatus. !he distance between DNAbands of di$erent lengths is inSuencedby the percent agarose in the gel, withhigher percentages requiring longerrun times, sometimes days.

7nstead high percentage agarose gelsshould be run with a pulsed "eldelectrophoresis +%F8, or "eld

inversion electrophoresis.

%olyacrylamide


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y y

is used for separating proteins rangingin sie from E to >,222 Da due to the

uniform pore sie provided by thepolyacrylamide gel.

%ore sie is controlled by modulating

the concentrations of acrylamide andbis-acrylamide powder used in creatinga gel. are must be used when creatingthis type of gel, as acrylamide is apotent neurotoxin in its liquid andpowdered forms.

!raditional DNA sequencing techniques


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q g qsuch as Taxam-Olibert or 0anger methodsused polyacrylamide gels to separate DNAfragments di$ering by a single base-pair inlength so the sequence could be read.

Tost modern DNA separation methods nowuse agarose gels, except for particularly

small DNA fragments. 7t is currently mostoften used in the "eld of immunology andprotein analysis, often used to separatedi$erent proteins of isoforms of the same

protein into separate bands. !hese can betransferred into a nitrocelluloseor %/DF membrane to be probed withantibodies and corresponding marers, such

as in a western blot.

0tarch


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0tarch

%artially hydrolysed potato starch

maes for another non-toxicmedium for proteinelectrophoresis. !he gels areslightly more opaque than

acrylamide or agarose. Non-denatured proteins can beseparated according to charge and

sie. !hey are visualised usingNapthal *lac or Amido *lacstaining. !ypical starch gelconcentrations are E3 to :23.

A*.antage% o- Gel


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A*.antage% o- GelElect!oho!e%i%

7mproved Diagnosis-Tedical laboratories use electrophoresis todiagnosis blood disorders.

0implicity 8lectrophoresis

-is a fast and easy technique

Kow-ost Taterial

-!he material necessary to perform

electrophoresis costs little and is easy to prepare eliability

-(f all the bene"ts electrophoresis o$ers,reliability is the most essential

Di%a*.antage% o- Gel


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Di%a*.antage% o- GelElect!oho!e%i%

8lectrophoresis ;as Kimited 0ample Analysis- 8lectrophoresis is speci"c to whatever tissueyouGve sampled.

8lectrophoresis Teasurements Are Not %recise-Oel electrophoresis can e$ectively separatesimilar proteins with di$erent weight

(nly ertain Tolecules an *e /isualied

-8lectrophoresis is excellent at separating andidentifying medium- to large-sied biomolecules.


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!han Hou ForKisteningIII

dna fingerprinting and gel electrophoresis

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