RAPDRandom Amplified polymorphic DNA.

Presented by: Abhi Giri M.sc-II (Oceanography) Sem – III / Paper – II

Guided by: Mrs S. P. Jayani (M.sc, DMLT)Head of Department

R.K.Talreja college, UNR-3

Cont

ents

• A brief History of RAPD.• An Introduction to RAPD.• HOW? It works.• Differences between genomes using RAPD.• Interpreting RAPD banding patterns.• Procedure / Protocol.• Advantages.• Disadvantages.• Summary.• Conclusions.• References.

Markers

Markers :- Any trait that can be identified with confidence & relative case and can be followed in a mapping population is called as marker.

Genetic marker :- Genetic marker is a specific location on a chromosome that is defined by a naked eye polymorphism as differences in electrophoretic mobility of specific proteins, or as differences in specific DNA sequence.

There are three types of markers namely:-• MORPHOLOGICAL MARKER• BIOCHEMICAL MARKER MOLECULAR MARKER

History • Shortly after Kary Mullis invented PCR it was realized that

short primer would bind several locations in a genome & thus could multiple fragments.

• Williams et.al (1990) developed RAPD a technique used very short 10 base primers to generate random fragments from template DNAs

• RAPD fragments can be separated & used as genetic markers or a kind of DNA fingerprint.

• Techniques related to RAPD include:• DNA Amplification Fingerprinting (DAF) - Caetano-Anolles et

al. (1991) uses very short (eight nucleotide long) primers• Arbitrary Primed PCR (AP-PCR) - Welsh and McClelland

(1990) uses longer primers, but lowers primer annealing stringency to get priming at many sites

INTRODUCTION• RAPD markers are decamer DNA fragments.• RAPD is a type of PCR reaction, Segments amplified are

Random.• No knowledge of DNA sequence required. Hence a

popular method.• In recent years, RAPD is used to Characterize, & Trace,

the phylogeny of diverse plant & animal species.• Identical 10-mer primer will or will not amplify a

segment of DNA, depending on positions that are complementary to the primer sequence.

How it Works ?• The principle is that, a single, short oligonucleotide primer,

which binds to many different loci, is used to amplify random sequences from a complex DNA template.

• This means that the amplified fragment generated by PCR depends on the length and size of both the primer and the target genome.

• These amplified products (of up to 3.0 kb) are usually separated on agarose gels (1.5-2.0%) and visualised by ethidium bromide staining.

• Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites.

• Recently, sequence characterised amplified regions (SCARs) analysis of RAPD polymorphisms showed that one cause of RAPD polymorphisms is chromosomal rearrangements such as insertions/deletions.

• In order for PCR to occur: 1) the primers must anneal in a particular orientation (such that they point towards each other) and, 2) they must anneal within a reasonable distance of one another.

FINDING DIFFERENCES BETWEEN GENOMESUSING RAPD ANALYSIS

• Consider the Figure 2 (genome A). If another DNA template (genome B) was obtained from a different (yet related) source, there would probably be some differences in the DNA sequence of the two templates. Suppose there was a change in sequence at primer annealing site #2

Figure 2 (genome A)

Figure 3 (genome B)

• Genome A and B can represent genomic DNA from two individuals in the same species or possibly from two different species.

• Certain portions of genomic DNA tend to much conserved (very little variation) while other portions tend to vary greatly among individuals within a species or among different species.

• The trick in RAPD PCR analysis is to:1. Find those sequences which have justenough variation to allow us to detect differences among the organisms that we are studying.2. find the right PCR primers which will allow us to detect sequence differences.

(genomes A and B) on a agarose gel

INTERPRETING RAPD BANDING PATTERNS• Each gel is analysed by scoring the present (1) or absent (0)

polymorphic bands in individual lanes. The scoring can be done based on the banding profiles which is clear and transparent (Fig. 5a) otherwise the scoring is very difficult (Fig. 5b).

(Fig. 5a) (Fig. 5b)

• Criteria for selecting scoring bands: 1) reproducibility—need to repeat experiments. 2) thickness 3) size and, 4) expected segregation observed in a mapping population.

• DNA polymorphism among individuals can be due to: 1) mismatches at the primer site. 2) appearance of a new primer site and, 3) length of the amplified region between primer sites.

• The NTSYS-pc software ver. 2.02 is used to estimate genetic similarities with the Jaccard’s coefficient.

Components of a PCR and RAPD Reactions

RAPD1. Buffer (containing Mg++) - usually

high Mg++ concentrations are used lowering annealing stringency

2. Template DNA3. 1 short primer (10 bases) not

known to anneal to any specific part of the template DNA

4. dNTPs5. Taq DNA Polymerase (or another

thermally stable DNA polymerase)

PCR1. Buffer (containing Mg++)

2. Template DNA3. 2 Primers that flank the

fragment of DNA to be amplified

4. dNTPs5. Taq DNA Polymerase (or

another thermally stable DNA polymerase)

Proc

edur

e RAPD involves following steps:-

• 1.The DNA of a selected species is isolated. -quality influences the outcome of the PCR -high molecular weight. -No impure template -less amount of RNA

• 2. An excess of selected decaoligonucleotide added. -10 base primer for PCR, only 1 primer per reaction - Short primer bind randomly on the chromosome.

• 3.This mixture is kept in a PCR equipment and is subjected to repeated cycles of DNA denaturation-renaturation-DNA replication.

• 4.During this process, the decaoligonucleotide will pair with the homologous sequence present at different locations in the DNA.

• 5.DNA replication extend the decaoligonucleotide and copy the sequence continuous with the sequence with which the selected oligonucleotide has paired.

P

roce

dure

co

nt.

• 6.The repeated cycles of denaturation-renaturation-DNA replication will amplify this sequence of DNA.

• 7.Amplification will takes place only of those regions of the genome that has the sequence complementary to the decaoligonucleotide at their both ends.

• 8. After several cycles of amplification the DNA is subjected to gel electrophoresis.

Initial denaturation at 94°C for 10 min. Denaturation at 94°C for 1 min. Annealing at 37-45°C for 1 min. Extension at 72°C for 1 min. Final extension at 72°C for 10 min. Cooling at 4°C.

• 9.The amplified DNA will form a distinct band. it is detected by ethidium bromide staining and visible fluorescence's under U.V. light

PCR Melting

94 oC

Melting

94 oC

AnnealingPrimers

50 oC

Extension

72 oCTe

mpe

ratu

re100

0

50

T i m e

5’3’3’5’

3’5’

5’

5’3’5’

3’5’5’

5’

5’

5’3’

3’5’

3’5’

5’3’5’3’

5’

RAPD

Melting94 oC

Tem

pera

ture

100

0

50

T i m e

5’3’

3’5’

RAPD

Melting94 oC

Tem

pera

ture

100

0

50

T i m e

3’5’

5’3’

Heat

RAPD

Melting94 oC

AnnealingPrimers

50 oC

Extension72 oC

Tem

pera

ture

100

0

50

T i m e

3’5’

5’3’5’

5’

Melting94 oC

RAPD

Melting94 oC

Melting94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture 100

0

50

T i m e

30x

3’5’

5’3’

Heat

Heat

5’

5’

5’

RAPD

Melting94 oC

Melting94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture 100

0

50

T i m e

30x

3’5’

5’3’5’

5’

5’

5’

5’

5’

RAPD

Melting94 oC

Melting94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture 100

0

50

T i m e

30x

3’5’

5’3’ 5’

5’5’

5’

5’

5’

Heat

Heat

Fragments of defined length

RAPD

Melting94 oC

Melting94 oCAnnealing

Primers50 oC

Extension72 oC

Tem

pera

ture

100

0

50

T i m e

30x

5’

5’

5’

5’

5’

5’

5’

5’

3’5’

5’3’ 5’

5’

Primers

• Primers are commercially available from various source (ex.. Opéron Technologies Inc.,California; Biosciences, Bangalore; Euro Finns, Bangalore; GCC Biotech, Kolkata).

prot

oco

l Isolation of DNA

Keep the tubes in PCR thermocycler

Denature the DNA (94°C,1 min

DNA strands separatedDecaoligonucleotide enzyme, primer, Taq DNA

polymerase,

Annealing of primer (36°C,2 min

Primer annealed to template DNA strands

DNA synthesis (72°C, 1.5 min

Prot

ocol

co

nt.

Complementary strand synthesis

35 to 45 cycles

Amplified products separated by gel electrophoresis

Bands detected by Ethidium bromide staining

RAPD

Template DNA

Primer binds to many locations on the template DNA

Only when primer binding sites are close and oriented in opposite direction so the primers point toward each other will amplification take place

RAPD

Template DNA

Primers point away from each other, so amplification won’t happen

RAPD

Template DNA

Primers point in the same direction, so amplification won’t happen

RAPD

Template DNA

Primers too far apart, so amplification won’t happen > 2,000 bases

RAPD

Template DNA

100 - 1,500 bases

Primers are just the right distance apart, so fragment is amplified

MM 2 3 4 5 6 7 8 9 10

Separated RAPD Fragments4mM MgCl2

1.2 U Taq5 pM OPA-16

4mM MgCl2

0.6 U Taq10 pM OPA-16

2mM MgCl2

1.2 U Taq10 pM OPA-16

Normal concentrations are shown in green text. M = A size standard

Lowering Magnesium ion concentration results in loss of the largest fragment visible in lanes 2-7

RAPD reactions were run in groups of 3 using the same template and primer, but varying Magnesium, polymerase and primer concentrations

Fig. no. 1 diagrammatic view of RAPD

Advantages• It requires no DNA probes and sequence information for the

design of specific primers.• It involves no blotting or hybridisation steps, hence, it is quick,

simple and efficient.• It requires only small amounts of DNA (about 10 ng per reaction)

and the procedure can be automated.• High number of fragments.• Arbitrary primers are easily purchased.• Unit costs per assay are low compared to other marker

technologies.

Disadvantages• Nearly all RAPD markers are dominant,• PCR is an enzymatic reaction, therefore, the quality and

concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome.

• Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product.

• Lack of a prior knowledge on the identity of the amplification products.

• Problems with reproducibility.• Problems of co-migration.

DEVELOPING LOCUS-SPECIFIC, CO-DOMINANTMARKERS FROM RAPDs

• The polymorphic RAPD marker band is isolated from the gel.

• It is amplified in the PCR reaction. The PCR product is cloned and sequenced.

• New longer and specific primers are designed for the DNA sequence, which is called the sequenced characterized amplified region marker (SCAR).

Applications • genetic diversity/polymorphism,• germplasm characterization,• genetic structure of populations,• hybrid purity,• genome mapping,• developing genetic markers linked to a trait in question,• population and evolutionary genetics,• plant and animal breeding,• animal-plant-microbe interactions,• pesticide/herbicide resistance.

Sum

mar

y• RAPD is a lab technique used to amplify unknown(random) DNA

segments

• It is a technique firstly DNA is isolated, which is then treated with decaoliganucleotide enzymes it act as a restriction enzymes which is used to cleave a short ten nucleotide segments of DNA.

• Then mixture is taken to PCR equipment and the process of DNA denaturation and the annealing of primer occcurs, then primer extension takes place for 35 to 45 cycles.

• DNA hybridizaion occurs at some segment of DNA amplification occurs at a particular site.

• DNA is subjected to gel electrophoresis,the amplified DNA will form distinct band detected by ethidium bromide staining and visible fluorescence’s under U.V.light

Conclusion • RAPD markers exhibit reasonable speed, cost and

efficiency compared with other methods and,• RAPD can be done in a moderate laboratory. Therefore,

despite its reproducibility problem, it will probably be important until better techniques are developed in terms of cost, time and labour.

ReferenceS

BOOK AUTHOR PUBLICATION YEARBOOK OF BIOTECHNOLOGY

SATYANARAYANA U. UPALLA INTERLINK 2010

BIOTECHNOLOGY Susan R. Barnum Vikas Publishing House 2010

BASIC BIOTECHNOLOGY,2nd Edition

Colin Rateledge and Bjorn Kristiansen

CambridgeUniv. Press 2008

MOLECULAR BIOLOGY Welsh J & McClelland MCambridgeUniv. Press 1990

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YOU