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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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