Berti Rapd

7/25/2019 Berti Rapd

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RAPDRandomly Amplified Polymorphic

DNA


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PCR AMPLIFICATION


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PCR APPLICATIONS

MOLECULAR BIOLOGY

ENVIRONMENTAL SCIENCE

FORENSIC/MEDICAL SCIENCE

BIOTECHNOLOGY

GENETICS

GENE CLONING

MICROBIOLOGY


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DNA STRUCTURE

SUGAR-PHOSPHATE BACKBONE

A=ADENINE

T=THYMINE

C=CYTOSINE

G=GUANINE


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PCR REQUIREMENTS

DNA

PRIMERS

dATP, dTTP, dCTP, dGTP

POLYMERASES


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RAPD PCR

RANDOM

AMPLIFIED

POLYMORPHIC

DNA


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RAPD PRIMERS

1 NUCLEOTIDES LONG

ARBITRARY SEQUENCE

BINDS AT MULTIPLE SEQUENCES ALONG

DNA

NON-SPECIFIC BINDING

ACCURACY REQUIRES AT LEAST 1

PRIMERS


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RAPD

- a method based on PCR developed in 1990.

- RAPD is different from conventional PCR as

it needs one primer for amplification. The

size of primer is normally short 10

n!cleotides"# and therefore# less specific.- the primers can be desi$ned %itho!t the

e&perimenter havin$ any $enetic information

for the or$anism bein$ tested.

- more than '000 different RAPD primers can

be available commercially.


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RAPD

- (enomic D)A normally has complimentary

se*!ences to RAPD primers at manylocations.

-+f t%o of these locations are close to each

other ,000bp"# and the se*!ences are inopposite orientation# the amplification %ill be

established. This amplified re$ion is said as a

RAPD loc!s.

-)ormally# a fe% -'0" loci can be amplifiedby one sin$le RAPD primer.


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RAPD

a" chan$e of se*!ence at primer annealin$site in the $enomic D)A

b" deletion of primer annealin$ site in the

$enomic D)A

c" lar$e insertion in bet%een t%o primer

annealin$ sites

ariation D)A detected by RAPD is d!e to

the loss of RAPD loci. The loss of RAPD

loci is ca!sed by/


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RAPD

ilver-stained polyacrylamide $el sho%in$ threedistinct RAPD profiles $enerated by primer P213

for Haemophilus ducreyiisolates from Tanzania#

ene$al# Thailand# 2!rope# and )orth America


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456(7

T2T 8R

8RA(52)T

8 +5+6AR5+6+T7 +)

RAPD

PR8+62


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RAPD

- RAPD mar:er is a dominant mar:er.- Presence of a D)A band is dominant;

absence of a D)A band is recessive.

- D)A bands of different sizes are

ass!med to be amplified prod!cts from

different RAPD loci.


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Modifications of RAPD

Techni*!es similar to RAPD/

AP-PCR

DA5D

+R


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AP-PCR

- AP-PCR Arbitrary Primed PCR".- similar to RAPD.

- involves t%o cycles of lo%-strin$ency

amplification# follo%ed by cycles

cond!cted at hi$her strin$ency# !sin$

primer of arbitrary se*!ence.


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AP-PCR

- the len$th of primers is '0-< n!cleotideslon$.

- the primers !sed incl!de the =niversal

51 se*!encin$ primer# the 51 reverse

se*!encin$ primer and the T se*!encin$primer.


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DAMD

- DA5D Directed Amplification of5inisatellite Re$ion D)A"

- techni*!e for detectin$ polymorphisms

!sin$ )TR core se*!ences as primers for

PCR


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ISSR

- +R +nter-imple e*!ence Repeat".- A PCR-based molec!lar mar:er assay of

$enomic se*!ence lyin$ bet%een ad>acent

microsatellites Rs". Primers carryin$#

at their ?-end# se*!ence complementaryto the repeat !nit of the microsatellite %ill

amplify this $enomic D)A.


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Criticism in RAPD

- lac: of reprod!cibility.- RAPD bandin$ patterns prone to/

i" D)A template concentration and *!ality

ii" Different Ta* D)A polymerasesiii" Different PCR machines or related

e*!ipment !sed in cond!ctin$ PCR.


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(enetic diversity parameters

Percenta$e of polymorphic loci hannon diversity inde H 4 @ ni@1 - i ln i

(enetic similarity# 8 8 @ 'm&y m& B my"

(enetic distance# 1-8


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ISOLASI DNA NYAMUK

AMPLIFIKASI


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Analisis elektroforesis


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