methods of dna analysis

8/14/2019 Methods of DNA Analysis

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Methods of DNA analysisMethods of DNA analysis


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Southern Blotting: Gel TransferSouthern Blotting: Gel Transfer


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Gel-transfer hybridizationor SouthernGel-transfer hybridizationor Southernblottingis used to detect specific DNAblottingis used to detect specific DNA

fragmentsfragments. (A) The mixture of double-stranded. (A) The mixture of double-stranded

DNA fragments generated by restriction nucleaseDNA fragments generated by restriction nuclease

treatment of DNA is separated according to lengthtreatment of DNA is separated according to length

by electrophoresisby electrophoresis


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A sheet of either nitrocellulose paper or nylonA sheet of either nitrocellulose paper or nylon

paper is laid over the gel, and the separatedpaper is laid over the gel, and the separatedDNA fragments are transferred to the sheet byDNA fragments are transferred to the sheet by

blotting. The gel is supported on a layer ofblotting. The gel is supported on a layer of

sponge in a bath of alkali solution, and thesponge in a bath of alkali solution, and the

buffer is sucked through the gel and thebuffer is sucked through the gel and the

nitrocellulose paper by paper towels stackednitrocellulose paper by paper towels stacked

on top of the nitrocellulose.on top of the nitrocellulose.


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As the buffer is sucked through, it denaturesAs the buffer is sucked through, it denatures

the DNA and transfers the single-strandedthe DNA and transfers the single-strandedfragments from the gel to the surface of thefragments from the gel to the surface of the

nitrocellulose sheet, where they adhere firmly.nitrocellulose sheet, where they adhere firmly.


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This transfer is necessary to keep the DNAThis transfer is necessary to keep the DNAfirmly in place while the hybridizationfirmly in place while the hybridization

procedure (D) is carried out. (C) Theprocedure (D) is carried out. (C) The

nitrocellulose sheet is carefully peeled off thenitrocellulose sheet is carefully peeled off thegel. (D)gel. (D)


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The sheet containing the bound single-The sheet containing the bound single-

stranded DNA fragments is placed in a sealedstranded DNA fragments is placed in a sealed

plastic bag together with buffer containing aplastic bag together with buffer containing a

radioactively labeled DNA probe specific forradioactively labeled DNA probe specific forthe required DNA sequencethe required DNA sequence


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The nitrocellulose sheet is carefully peeled offThe nitrocellulose sheet is carefully peeled off

the gel. (D) The sheet containing the boundthe gel. (D) The sheet containing the boundsingle-stranded DNA fragments is placed in asingle-stranded DNA fragments is placed in a

sealed plastic bag together with buffersealed plastic bag together with buffer

containing a radioactively labeled DNA probecontaining a radioactively labeled DNA probespecific for the required DNA sequence.specific for the required DNA sequence.


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The sheet is exposed for a prolonged period toThe sheet is exposed for a prolonged period to

the probe under conditions favoringthe probe under conditions favoring

hybridization. (E) The sheet is removed fromhybridization. (E) The sheet is removed from

the bag and washed thoroughly, so that onlythe bag and washed thoroughly, so that only

probe molecules that have hybridized to theprobe molecules that have hybridized to theDNA on the paper remain attached.DNA on the paper remain attached.


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After autoradiography, the DNA that hasAfter autoradiography, the DNA that has

hybridized to the labeled probe will show uphybridized to the labeled probe will show up

as bands on the autoradiograph. An adaptationas bands on the autoradiograph. An adaptation

of this technique to detect specific sequencesof this technique to detect specific sequences

in RNA is calledin RNA is calledNorthern blottingNorthern blotting..


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In this case mRNA molecules areIn this case mRNA molecules areelectrophoresed through the gel and the probeelectrophoresed through the gel and the probe

is usually a single-stranded DNA moleculeis usually a single-stranded DNA molecule


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DNA fingerprintingDNA fingerprinting


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Like the fingerprints that came into use by detectivesLike the fingerprints that came into use by detectives

and police labs during the 1930s, each person has aand police labs during the 1930s, each person has aunique DNA fingerprintunique DNA fingerprint.. Unlike a conventionalUnlike a conventional

fingerprint that occurs only on the fingertips and canfingerprint that occurs only on the fingertips and can

be altered by surgery, abe altered by surgery, a DNA fingerprintDNA fingerprint is the sameis the same

for every cell, tissue, and organ of a person. It cannotfor every cell, tissue, and organ of a person. It cannotbe altered by any known treatment. Consequently,be altered by any known treatment. Consequently,

DNA fingerprinting is rapidly becoming the primaryDNA fingerprinting is rapidly becoming the primary

method for identifying and distinguishing amongmethod for identifying and distinguishing among

individual human beings.individual human beings.


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DNA fingerprinting is a laboratory procedureDNA fingerprinting is a laboratory procedurethat requires six stepsthat requires six steps


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Isolation of DNA.Isolation of DNA. DNA must be recovered from the cells orDNA must be recovered from the cells or

tissues of the body. Only a small amount oftissues of the body. Only a small amount of

tissue, like blood, hair, or skin, is needed. Fortissue, like blood, hair, or skin, is needed. For

example, the amount of DNA found at the rootexample, the amount of DNA found at the root

of one hair is usually sufficient.of one hair is usually sufficient.


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Cutting, sizing, and sorting.Cutting, sizing, and sorting. SpecialSpecial

enzymes calledenzymes called restriction enzymesrestriction enzymes are usedare used

to cut the DNA at specific places. Forto cut the DNA at specific places. For

example, an enzyme called EcoR1, found inexample, an enzyme called EcoR1, found in

bacteria, will cut DNA only when thebacteria, will cut DNA only when thesequence GAATTC occurs.sequence GAATTC occurs.


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The DNA pieces are sorted according to sizeThe DNA pieces are sorted according to size

by a sieving technique calledby a sieving technique called electrophoresiselectrophoresis..The DNA pieces are passed through a gelThe DNA pieces are passed through a gel

made from seaweed agarose (a jelly-likemade from seaweed agarose (a jelly-like

product made from seaweed). This techniqueproduct made from seaweed). This techniqueis the DNA equivalent of screening sandis the DNA equivalent of screening sand

through progressively finer mesh screens tothrough progressively finer mesh screens to

determine particle sizesdetermine particle sizes


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3) Transfer of DNA to nylon.3) Transfer of DNA to nylon. TheThe

distribution of DNA pieces is transferred to adistribution of DNA pieces is transferred to anylon sheet by placing the sheet on the gel andnylon sheet by placing the sheet on the gel and

soaking them overnight.soaking them overnight.


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4-5) Probing.4-5) Probing. Adding radioactive or coloredAdding radioactive or colored

probes to the nylon sheet produces a patternprobes to the nylon sheet produces a patterncalled the DNA fingerprint. Each probecalled the DNA fingerprint. Each probe

typically sticks in only one or two specifictypically sticks in only one or two specific

places on the nylon sheet.places on the nylon sheet.


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6) DNA fingerprint.6) DNA fingerprint. The final DNAThe final DNAfingerprint is built by using several probes (5-fingerprint is built by using several probes (5-

10 or more) simultaneously. It resembles the10 or more) simultaneously. It resembles the

bar codes used by grocery store scanners.bar codes used by grocery store scanners.


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Uses of DNA FingerprintsUses of DNA Fingerprints

DNA fingerprints are useful in several areas ofDNA fingerprints are useful in several areas of

society. They are used by professionals insociety. They are used by professionals in

human health and the justice system.human health and the justice system.


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Diagnosis of inherited disordersDiagnosis of inherited disorders

DNA fingerprinting is used to diagnoseDNA fingerprinting is used to diagnose

inherited disorders in both prenatal andinherited disorders in both prenatal and

newborn babies in hospitals around the world.newborn babies in hospitals around the world.

These disorders may include cystic fibrosis,These disorders may include cystic fibrosis,

hemophilia, Huntington's disease, familialhemophilia, Huntington's disease, familial

Alzheimer's, sickle cell anemia, thalassemia,Alzheimer's, sickle cell anemia, thalassemia,

and many others.and many others.


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Early detection of such disorders enables theEarly detection of such disorders enables the

medical staff to prepare themselves and themedical staff to prepare themselves and theparents for proper treatment of the child. Inparents for proper treatment of the child. Insome programs, genetic counselors use DNAsome programs, genetic counselors use DNAfingerprint information to help prospectivefingerprint information to help prospective

parents understand the risk of having anparents understand the risk of having anaffected child. In other programs, prospectiveaffected child. In other programs, prospectiveparents use DNA fingerprint information inparents use DNA fingerprint information intheir decisions concerning affectedtheir decisions concerning affectedpregnanciespregnancies


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Developing cures for inheritedDeveloping cures for inherited

disordersdisorders Research programs to locate inherited disorders onResearch programs to locate inherited disorders on

the chromosomes depend on the informationthe chromosomes depend on the information

contained in DNA fingerprints. By studying the DNAcontained in DNA fingerprints. By studying the DNA

fingerprints of relatives who have a history of somefingerprints of relatives who have a history of someparticular disorder, or by comparing large groups ofparticular disorder, or by comparing large groups of

people with and without the disorder, it is possible topeople with and without the disorder, it is possible to

identify DNA patterns associated with the disease inidentify DNA patterns associated with the disease in

question. This work is a necessary first step inquestion. This work is a necessary first step indesigning an eventual genetic cure for thesedesigning an eventual genetic cure for these

disorders.disorders.


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Another important use of DNA fingerprints inAnother important use of DNA fingerprints inthe court system is to establish paternity inthe court system is to establish paternity in

custody and child support litigation. In thesecustody and child support litigation. In these

applications, DNA fingerprints bring anapplications, DNA fingerprints bring anunprecedented, nearly perfect accuracy to theunprecedented, nearly perfect accuracy to the

determinationdetermination


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Personal identificationPersonal identification

Because every organ or tissue of an individualBecause every organ or tissue of an individual

contains the same DNA fingerprint, the U.S. armedcontains the same DNA fingerprint, the U.S. armed

services have just begun a program to collect DNAservices have just begun a program to collect DNA

fingerprints from all personnel for use later, in casefingerprints from all personnel for use later, in casethey are needed to identify casualties or personsthey are needed to identify casualties or persons

missing in action. The DNA method will be farmissing in action. The DNA method will be far

superior to the dogtags, dental records, and bloodsuperior to the dogtags, dental records, and blood

typing strategies currently in use.typing strategies currently in use.


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All primates have ridged skin, and it can alsoAll primates have ridged skin, and it can also

be found on the paws of certain mammals andbe found on the paws of certain mammals and

on the tails of some monkey species. Inon the tails of some monkey species. In

humans and animals,humans and animals, dermatoglyphsdermatoglyphs areare

present on fingers, palms, toes, and soles, andpresent on fingers, palms, toes, and soles, and

give insight into a critical period ofgive insight into a critical period ofembryogenesisembryogenesis, between 4 weeks and 5, between 4 weeks and 5

months, when the architecture of the majormonths, when the architecture of the major

organ systems is developing.organ systems is developing.
http://en.wikipedia.org/wiki/Embryogenesishttp://en.wikipedia.org/wiki/Embryogenesishttp://en.wikipedia.org/wiki/Embryogenesis


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Triradius: point of convergence of ridges fromTriradius: point of convergence of ridges from

3 different directions. Normally, there is:3 different directions. Normally, there is: 1 axial triradius: normally in t, close to the1 axial triradius: normally in t, close to the

wrist.wrist.

4 subdigital triradii (a.b.c.d.).4 subdigital triradii (a.b.c.d.).


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On the pad of the distal phalanx, sometimes onOn the pad of the distal phalanx, sometimes on

thenar or hypothenar eminences, are triradii,thenar or hypothenar eminences, are triradii,

accompanied with the following patterns:accompanied with the following patterns: worl: 2 triradii.worl: 2 triradii.

loops and equivalents (ulnar or radial orientated): 1loops and equivalents (ulnar or radial orientated): 1

triradius.triradius.

arches: 0 triradius.arches: 0 triradius.


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From each palmar triradius a, b, c, d, and t, isFrom each palmar triradius a, b, c, d, and t, is

drawn the 3 lines separating the ridges at thisdrawn the 3 lines separating the ridges at this

convergence point. The longest is the mainconvergence point. The longest is the main

line (-- A B C D & T), ending at a side of theline (-- A B C D & T), ending at a side of thepalm numbered from 1 to 14palm numbered from 1 to 14


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T normally ends in 13.T normally ends in 13.

transversality index = A+B+C+D = 27 on thetransversality index = A+B+C+D = 27 on the

FigureFigure


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Genetic disordersGenetic disorders

Unusual dermatoglyphic patterns often relateUnusual dermatoglyphic patterns often relate

to genetic disordersto genetic disorders

One study of foetuses with chromosomalOne study of foetuses with chromosomal

abnormalities showed that the dermatoglyphicabnormalities showed that the dermatoglyphic

patterns were delayed by more than two weekspatterns were delayed by more than two weeks

Trisomy 21 (Trisomy 21 (Down syndromeDown syndrome): People with Down): People with Down
http://en.wikipedia.org/wiki/Down_syndromehttp://en.wikipedia.org/wiki/Down_syndromehttp://en.wikipedia.org/wiki/Down_syndrome


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Trisomy 21 (Trisomy 21 (Down syndromeDown syndrome): People with Down): People with Downsyndrome have mainlysyndrome have mainly ulnar loopsulnar loops, and a, and asignificantly different angle between the triradia a, tsignificantly different angle between the triradia a, tand d (the 'adt angle').and d (the 'adt angle').
http://en.wikipedia.org/wiki/Down_syndromehttp://en.wikipedia.org/wiki/Down_syndromehttp://en.wikipedia.org/wiki/Ulnar_loophttp://en.wikipedia.org/wiki/Ulnar_loophttp://en.wikipedia.org/wiki/Ulnar_loophttp://en.wikipedia.org/wiki/Down_syndrome


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Other differences often include aOther differences often include a

single transverse palmar creasesingle transverse palmar crease ("Simian line")("Simian line")(in 50%), and patterns in the hypothenar and(in 50%), and patterns in the hypothenar and

interdigital areas, lower ridge counts alonginterdigital areas, lower ridge counts along

digital midlines, especially in little fingers,digital midlines, especially in little fingers,which corresponds to finger shortening in thosewhich corresponds to finger shortening in those

with Down's syndromewith Down's syndrome
http://en.wikipedia.org/wiki/Single_transverse_palmar_creasehttp://en.wikipedia.org/wiki/Single_transverse_palmar_creasehttp://en.wikipedia.org/wiki/Single_transverse_palmar_crease


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There is less variation in dermatoglyphicThere is less variation in dermatoglyphic

patterns between people with Down syndromepatterns between people with Down syndrome

than between controls,than between controls, and dermatoglyphicand dermatoglyphic

patterns can be used to determine correlationspatterns can be used to determine correlations

with congenital heart defects in individualswith congenital heart defects in individuals

with Down syndrome by examining the leftwith Down syndrome by examining the lefthand digit ridge count minus the right handhand digit ridge count minus the right hand

digit ridge count, and the number of ridges ondigit ridge count, and the number of ridges on

the fifth digit of the left handthe fifth digit of the left hand


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Turner syndromeTurner syndrome: Predominance of whorls,: Predominance of whorls,

although the pattern frequency depends on thealthough the pattern frequency depends on the

particular chromosomal abnormalityparticular chromosomal abnormality
http://en.wikipedia.org/wiki/Turners_syndromehttp://en.wikipedia.org/wiki/Turners_syndromehttp://en.wikipedia.org/wiki/Turners_syndrome


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47, XXY (47, XXY (Klinefelter's syndromeKlinefelter's syndrome): Excess of): Excess of

arches on digit 1, more frequent ulnar loops onarches on digit 1, more frequent ulnar loops ondigit 2, overall fewer whorls, lower ridgedigit 2, overall fewer whorls, lower ridge

counts for loops and whorls as compared withcounts for loops and whorls as compared with

controls, and significant reduction of the totalcontrols, and significant reduction of the totalfinger ridge countfinger ridge count
http://en.wikipedia.org/wiki/Klinefelter%27s_syndromehttp://en.wikipedia.org/wiki/Klinefelter%27s_syndromehttp://en.wikipedia.org/wiki/Klinefelter%27s_syndrome


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Trisomy 13 (Trisomy 13 (Patau syndromePatau syndrome): Excess of): Excess of

arches on fingertips andarches on fingertips and

single transverse palmar creasessingle transverse palmar creases in 60%.in 60%.
http://en.wikipedia.org/wiki/Patau_syndromehttp://en.wikipedia.org/wiki/Patau_syndromehttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Patau_syndrome


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Trisomy 18 (Trisomy 18 (Edward's syndromeEdward's syndrome) 6 - 10 arches) 6 - 10 arches

on fingertips andon fingertips and

single transverse palmar creasessingle transverse palmar creases in 30%.in 30%.
http://en.wikipedia.org/wiki/Edward%27s_syndromehttp://en.wikipedia.org/wiki/Edward%27s_syndromehttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Edward%27s_syndrome


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Cri du chatCri du chat (5p-): Excess of arches on(5p-): Excess of arches onfingertips andfingertips and single transverse palmar creasessingle transverse palmar creases

in 90%.in 90%.
http://en.wikipedia.org/wiki/Cri_du_chathttp://en.wikipedia.org/wiki/Cri_du_chathttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Single_palmar_creasehttp://en.wikipedia.org/wiki/Cri_du_chat


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Inborn blindness : Dermatoglyphic analysis ofInborn blindness : Dermatoglyphic analysis of

finger tip print patterns of blind children fromfinger tip print patterns of blind children fromBangaloreBangalore
http://go2.wordpress.com/?id=725X1342&site=palmistryreport.wordpress.com&url=http%3A%2F%2Fwww.handresearch.com%2Fnews%2Fbarack-obama-the-hand-of-power.htm


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