MOLECULAR MARKERS: RFLP,AFLP AND DATA ANALYSIS

ASHWIN JAYALEID no-PALB1222

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RFLP= Restriction fragment length polymorphism

Different fragment lengths of base pairs that result from cutting a DNA molecule with restriction enzymes

Refers to variation in restriction sites between individuals in a population

These are extremely useful and valuable for geneticists.

On average two individuals (humans) vary at 1 in 1000 bp

The human genome is 3x109 bp

This means that they will differ in more than 3 million bp.

By chance these changes will create or destroy the recognition sites for

Restriction enzymes

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The DNA sample is broken into pieces (digested)

by restriction enzymes and the resulting restriction

fragments are separated according to their lengths

by gel electrophoresis

It refers to a difference between samples

of homologous DNA molecules that come from

differing locations of restriction enzyme sites.

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mechanism

1. DNA is cut into fragments by the restriction enzyme

2. Fragments are separated into bands by electrophoresis

3. DNA bands are transferred to a nylon membrane

4. A radioactive DNA probe is added to the membrane where it binds to specific fragments

5. X-ray film is placed on top of the nylon membrane to detect radioactive pattern

6. Xray film is developed.

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During RFLP typing restriction enzymes cut up fragments into hundreds of fragments- some containing repeating sequences from the DNA molecule

It is necessary to follow this procedure with gel Electrophoresis

Electrophoresis Once the DNA molecules have been cut up by

restriction enzymes the fragments have to be sorted out.

This is accomplished through electrophoresis Electrophoresis- a technique for separating molecules

through their migration on a support medium under the influence of an electrical potential

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DNA cut up from restriction enzymes is placed on a plate coated with a gel medium (usually agar or starch)

When the gel is subjected to an electric potential the DNA fragments migrate across the plate

The smaller fragments move quickly across the plate, so the fragments are separated by size

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Southern Blotting

Once the electrophoresis process is completed, the double stranded fragments of DNA are chemically treated so the strands separate from each other.

The fragments are then transferred to a nylon membrane in the same manner as one would transfer an ink line onto a blotter.

Transfer process is called southern blotting after its developer Edward Southern.

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Hybridization

To visualize the separated RFLPs, the nylon sheet is treated with radioactively labeled probes

containing a base sequence complementary to the RFLPs being identified.

Hybridization- the process of joining 2 complementary strands of DNA to form a double stranded

molecule.

Probes:- A molecular probe is a group of atoms or molecules attached to other molecules or cellular

structures and used in studying the properties of these molecules and structures.

Radioactive DNA or RNA sequences are used in molecular genetics to detect the presence of

a complementary sequence by molecular hybridization.

Types :a) genomic DNA probes.

b) c DNA probes.

c) RNA probes.

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summary

Portions of the DNA molecule contain sequences of bases that are repeated numerous times. These tandem repeats can distinguish one individual from another

Materials are separated by electrophoresis where the DNA is separated by electric current

A typical DNA fragment pattern shows 2 bands.

RFLP are codominant marker.

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T-REX: software for the processing and analysis of T-RFLP data

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AFLPAFLPAMPLIFIED FRAGMENT LENGTH AMPLIFIED FRAGMENT LENGTH

POLYMORPHISMPOLYMORPHISM

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AFLPAFLP

-A method based on PCR developed in 1995 by Zabeau, Vos and co workers.

- Involves the use of RFLP and PCR techniques.

- Compared with the widely used RFLP, AFLP is faster, less labour intensive and provide more information.

- An additional advantage over RAPD is their reproducibility.

-Powerful approach to detect polymorphism.

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AFLPAFLP

The AFLP technique is based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.

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AFLP

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AFLP is a technique in which differences in restriction fragments

are revealed by PCR, and this not for one locus but for a larger

number of loci in one reaction

In a first step the restriction fragments are generated by using two

different enzymes (a frequent tetra-cutter and a more rare

hexacutter)

Adapters are ligated to these fragments in order to have known

sequences for primer design

Selected fragments are amplified (to have between 50-150 bands

on the gel) and separated by polyacrylamide gel electrophoresis

(detection by autoradiography or fluorescence)

AFLPAFLP

Procedures in AFLP:

- Digestion

- Adaptor Ligation

- Amplification

- Electrophoresis

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Two different restriction endonucleases are used in digestion. One is 4-base cutter (MseI) and the other one is 6-base cutter (EcoRI).

DigestionDigestion

MseI 5’TTAA3’

EcoRI 5’GAATTC3’

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- Two different adaptors (short double stranded DNA sequences with sticky end) are ligated to the digested fragments.

- One adaptor will complement to the Msel cut end, the other will complement to the EcoRI cut end.

Adaptor LigationAdaptor Ligation

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AFLP

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AFLP

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Selected fragments are amplified (to have 50-150 bands on the gel) and separated by polyacrylamide gel electrophoresis (silver staining, autoradiography or fluorescence)

This selection is made by using longer primers: every extra nucleotide decreases the number of fragments by 1/4, so 2 extra nucleotides on each primer will amplify 1/256

By repeating this second amplification with other primer pairs (other selective nucleotides) a different subset of the genome is amplified.

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- polyacrylamide gel is used for separating DNA bands.

- Normally, 30-100 DNA bands can be detected by AFLP on polycrylamide gel.

ElectrophoresisElectrophoresis

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The main advantages of AFLP are No need for known sequences in the

genome High reproducibility Many loci are simultaneously analysed By changing the selective nucleotides a

different part of the genome (and thus different loci) can be analysed

Whole genome analysis is (theoretically) possible

The main disadvantage: complex procedure.

AFLP

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Polymorphism among 32 wheat samples revealed by AFLP

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- The number of DNA bands detected by AFLP is high. It can be reduced by adding selective bases (1-3 nucleotides) at the 3’-end of the PCR primers.

- one additional selective base on the primer can reduced the number of DNA bands 16 folds.

- three additional selective bases on the primer can reduce the number of DNA bands 4,000 folds.

Selective BasesSelective Bases

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- Dominant marker.

- DNA variation is detected by presence/absence of DNA bands due to:

a) presence/absence of restriction sites

b) additional bases (insertion) between two restriction sites are too large

Characteristics of AFLPCharacteristics of AFLP

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- higher reproducibility compared to RAPD.

- highly polymorphic.

AdvantagesAdvantages

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Analysis of AFLP data

Similarity (cluster analysis) NJ (Neighbor Joining) UPGMA (Unweighted Pair Group Method with Arithmetic

mean) AMOVA (Analysis of Molecular Variance)

Model-based Maximum likelihood Bayesian

Image Source: http://media.wiley.com/CurrentProtocols/BI/bi0603/bi0603-fig-0002-1-full.gif

Example of a sequence distance matrix

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AFLP Clustering Analysis

Source: Wikimedia Commons

Clustering Dendrogram Fragment Visualization

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BioNumerics software package (Applied Maths, Kortrijk, Belgium). 

AFLP Data Map with UPGMA dendogram from Urbanelli et al. (2007): “Distinguishing taxa in the Pleurotus eryngii (King Oyster Mushroom) complex using AFLPs”• 90 populations sampled• 94 AFLP loci scored

Photos: (Top) The New York Times (Bottom L) Wikimedia Commons (Bottom R) http://steinpilz.up.seesaa.net

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Thank you

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