random amplified polymorphic dna (rapd) - ipmb...

Random Amplified Polymorphic DNA (RAPD)

By: Jegan IYYATHURAI23-12-2010

IPMB- 1st Year

Contents

• The concept

• Principles of RAPD techniques and Protocol

• Application

• Advantages and Disadvantages

• Conclusion

1974 1990 1995

RFLP RAPD AFLP

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Evolution of DNA markers

RFLP- Restriction Fragment Length Polymorphism

RAPD- Restriction Amplified Polymorphic DNA

AFLP- Amplified Fragment Length Polymorphism

What is marker?

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Types of markers

Morphological

Biochemical Genetic or molecular

Chromosomal

Marker is a piece of

DNA molecule that is

associated with a

certain trait of a

organism

• Random Amplified Polymorphic DNA (RAPD) technique based on Polymerase Chain Reaction (PCR), which is most commenly used in molecular biolology technique to develop molecular marker.

• The primer size is normally 10 nucleotides

• The primer can design without any genetic information

• > 2000 different types of RAPD primers available

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

• RAPD locus (amplified region) not more than 3000 bp

• Normally 3 to 2O loci can amplify by one single primer

• The loss of RAPD loci is caused by

– Length between two primers

– Insertion of gene between two primer site

– Delition of primer annealing site in the genomic DNA

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

• Isolation of genomic DNA

• Purification of DNA

• PCR amplification

• Seperate the amplified products by

gel electroporesis

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

• The primers must anneal in a particular orientation

• The primers must anneal with a reasonable distance

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

1

2

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A B

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

1

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B

M # 1 # 2

Product A

Product B

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Calculating Genetic Distances from RAPD Data

Using software …..

• Population genetic variation or Genetic diversity

– Between- population variation

– Within- population variation

• Animal and Plant breeding

• Gene mapping

• Molecular polymorphism

– Protein variation

– DNA sequence variation

• Pre and postnatal diagnosis of disease

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

• Tiny amount of DNA are sufficient

• Arbitary primers easy to purchase

• Unit cost per assay are low

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Advantages of RAPDs

• Dominant

• Problems with reproductivity

• RAPD banding patterns prone to

– Genomic DNA quality and quantity

– Different brand of Taq polymerase enzyme

– Different PCR machine

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Disadvantages of RAPDs

• Title:

Comparison of RAPD and PCR-RFLP markers for classification and taxonomic studies of insects

• Objective:

Determine the phylogenic tree for three ants

• Materials and Methods:

– DNA extraction from three ants

– PCR amplification

– Run on agrose gel 2%

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Example 1

Camponotus maculatus

Cataglyphis bicolor

Monomorium pharoensis

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Results:

Example 1 (continued)

• Title:

Identification of Genomic Markers by RAPD-PCR Primer in

Leukemia Patients (Biotechnology 9 (2): 170-175, 2010)

• Objective:

To investigate DNA polymorphisms and detection of genomi

markers in various types of Leukemia

• Materials and methades– Isolation of DNA

– DNA amplification

– Gel electrophoresis- 1.5 % agerose

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Example: 2

• Results:

• Discussion:The fragment of 980 bp in the genomic DNA of CML, AML and ALL may use as a molecular marker for diagnosis and prognosis of leukemai

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Example: 2 (continued)

• The success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the requirement for cloning, sequencing or any other form of the molecular characterisation of the genome of the species.

Introduction- Concept- Prtocol- Applictions- Advantages and Disadvantages- Conclusion

Thank you !!