Download - DNA Fingerprinting and Gel Electrophoresis
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
1/58
DNAFINGERPRINTING
AND GELELECTROPHORESIS
Noynay, Rigil KentGuyha, Chuck !ail"illa!in, #e$e!ie DaleDingla%a, E!icka &inette #u$a$il, K!i%%hia &haeLini%, Shanyne
'!(i)ton*o, #ianne #ah*iel
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
2/58
- any of several similar techniques
for analysing and comparing DNA- used especially in lawenforcement to identify suspects
from hair, blood, semen, or otherbiological materials found at thescene of a violent crime
DNA +nge!!inting
o! DNA !o+ling
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
3/58
- it depends on the fact that notwo people, have exactly thesame DNA sequence, and thosesegments will be statistically
unique.Fingerprint
- very unliely that any twopeople would have exactly thesame DNA
information.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
4/58
!he test is used to determine
whether a family relationshipexists between two people, toidentify organisms causing adisease, and to solve crimes.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
5/58
!he procedure for creating aDNA "ngerprint consists of "rst
obtaining a sample of cells, suchas sin, hair, or blood cells,which contain DNA. !he DNA isextracted from the cells andpuri"ed.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
6/58
Poly$o!hi%$
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
7/58
A polymorphism is a clinicallyharmless DNA variation thatdoes not a$ect the phenotype.
At the molecular level,polymorphism is a variation in
nucleotide sequence from oneindividual to another.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
8/58
%olymorphisms often occur inthe intervening sequences thatdo not code for proteins. &Note'
(nly a few percent of the humangenome actually encodesproteins.)
All the common blood types,such as the A*( blood groupsystem , are genetic
polymorphisms.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
9/58
Tan*e$ !eeat%
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
10/58
%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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
11/58
!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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
12/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
13/58
7n either case, cleavage with anendonuclease results in fragmentsof lengths di$ering from thenormal, which can be detected byDNA hybridiation.
!he altered restriction site can beeither at the site of a disease-causing mutation or at a site somedistance from the mutation.
8xample ' 0icle cell anaemia.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
14/58
Alication o-DNA
Finge!!intin
g
)
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
15/58
'%e% o- DNA P!o+ling
DNA pro"ling isused to solvecrimes and
medicalproblems
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
16/58
C!i$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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
17/58
iological $ate!ial%
u%e* -o! DNA !o+ling*lood;air
0aliva0emen*ody tissue
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
18/58
DNA P!o+ling can %ol.ec!i$e%
!he pattern of the DNApro"le is then compared withthose of the victim and the
suspect.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
19/58
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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
20/58
E/a$le
A violent murder occurred.
!he forensics team retrieved ablood sample from the crimescene.
!hey prepared DNA pro"les ofthe blood sample, the victimand a suspect as follows'
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
21/58
0a% the %u%ect at thec!i$e %cene1
Suspects
Profile
Blood sample from
crime sceneVictims
profile
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
22/58
Sol.ing &e*icalP!o(le$%DNA pro"les can be used to determine
whether a particular person is the
parent of a child.
A child=s paternity +father andmaternity+mother can be determined.
!his information can be used in %aternity suits
7nheritance cases
7mmigration cases
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
23/58
E/a$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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
24/58
I% thi% $an the -athe! o- thechil*1
Mother Child Man
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
25/58
Fa$ou% ca%e%
7n >22>8liabeth;urley usedDNA pro"lingto prove that0teve *ing was
the father
of her childDamien
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
26/58
Fa$ou% Ca%e%
olin %itchfor was the "rstcriminal caught based on DNA"ngerprinting evidence.
;e was arrested in :1?@ forthe rape and murder of twogirls and was sentenced in:1??.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
27/58
Fa$ou% Ca%e%
(.. 0impson wascleared of a doublemurder charge in :11B
which relied heavily onDNA evidence.
!his case highlightedlab diCculties.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
28/58
S'STAGES OF
DNA
FINGERPRINTING
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
29/58
:' 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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
30/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
31/58
?' !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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
32/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
33/58
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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
34/58
*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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
35/58
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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
36/58
!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..
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
37/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
38/58
!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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
39/58
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$ordable and reliable technique
9. 7t can be conducted in a relatively short amount of time
B. Anyone at any age can be tested with this methodwithout any ma6or concerns
E. !here is a large variety of uses such as in legal claims,missing persons cases, identi"cation for the military, andpaternity and prenatal testing
@. !he technique has used since :1?B, maing it highlydeveloped and improved
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
40/58
Di%a*.antage% o- DNA Finge!!inting :. !he sample of DNA can easily be ruined during the
process of DNA "ngerprinting, causing the sample tobecome completely useless for testing
>. !he process itself is complex and tedious, and can giveresults that may be hard to interpret
9. !he test needs to be run on multiple samples, anumerous amount of times for ideal accuracy. ommonly,labs run each test twice with four samples.
B. %rivacy issues could occur if the information isnGt eptsecure at the lab. %ersonal information legally can only bereleased with a written order. !his personal information ifleaed, could potentially complicate insurance processes,health care and 6ob prospects for an individual
Alec #e4!ey%
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
41/58
Alec #e4!ey%
*orn 'Alec ohn
e$reys anuary 1, :1E2
(xford,(xfordshire, 8ngland,Lnited Mingdom
Age ' @E Hearsold
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
42/58
;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.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
43/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
44/58
- method of separation ofnanoparticles
- uses a gel as an anticonvectivemedium and
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
45/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
46/58
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$erent sied molecules form distinct bands on
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
47/58
Rhen separating proteins or small nucleicacids +DNA, NA or oligonucleotides the gel isusually composed of di$erent concentrations of
acrylamide and a cross-liner, producing di$erentsied mesh networs of polyacrylamide.
Rhen separating larger nucleic acids +greaterthan a few hundred bases, the preferred matrixis puri"ed agarose.
7n both cases, the gel forms a solid, yet porousmatrix.
Acrylamide - is a neurotoxin and must be handledusing appropriate safety precautions to avoidpoisoning.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
48/58
Agarose - composed of long unbranchedchains of uncharged carbohydrate without
cross lins resulting in a gel with largepores allowing for the separation ofmacromolecules and macromolecularcomplexes.
7f charges are not all uniform then,
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
49/58
7f charges are not all uniform then,the electrical "eld generated by theelectrophoresis procedure will a$ect
the species that have di$erentcharges and therefore will attractthe species according to their
charges being the opposite.
athode - negatively charged
- positively charged specieswill migrate towards it.Anode - positively charged
- negatively charged species
will mi rate towards it.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
50/58
Tye% o- Gel in Gel
Elect!oho!e%i%Agarose
- 7t is made from the naturalpolysaccharide polymers extractedfrom seaweed.
- are easily cast and handled comparedto other matrices, because the gelsetting is a physical rather thanchemical change.
0amples are also easily recovered
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
51/58
0amples are also easily recovered.After the experiment is "nished, theresulting gel can be stored in a plastic
bag in a refrigerator.
- do not have a uniform pore sie, but
are optimal for electrophoresis ofproteins that are larger than >22 Da.
Agarose gel electrophoresis can also be
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
52/58
Agarose gel electrophoresis can also beused for the separation of DNAfragments ranging from E2 base pair to
several megabases +millions of bases,the largest of which require specialiedapparatus. !he distance between DNAbands of di$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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
53/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
54/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
55/58
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
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
56/58
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$ers,reliability is the most essential
Di%a*.antage% o- Gel
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
57/58
Di%a*.antage% o- GelElect!oho!e%i%
8lectrophoresis ;as Kimited 0ample Analysis- 8lectrophoresis is speci"c to whatever tissueyouGve sampled.
8lectrophoresis Teasurements Are Not %recise-Oel electrophoresis can e$ectively separatesimilar proteins with di$erent weight
(nly ertain Tolecules an *e /isualied
-8lectrophoresis is excellent at separating andidentifying medium- to large-sied biomolecules.
-
8/20/2019 DNA Fingerprinting and Gel Electrophoresis
58/58
!han Hou ForKisteningIII