1 MOLECULAR MARKERS: RFLP,AFLP AND DATA ANALYSIS ASHW IN JAYALE ID no-
RFLP= Restriction fragment length polymorphism2 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
3 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.
mechanism4 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
5 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
6 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
Southern Blotting7 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.
Hybridization8 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.
summary9 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.
T-R X: software for the processing and E analysis of T-RFLP data10
11AFLPAMPLIFIED FRAGMENT LENGTHPOLYMORPHISM
AFLP12 -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.
AFLP13 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.
AFLP14 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)
Digestion Two different restriction endonucleases are used in digestion. One is 4-base cutter (MseI) and the other one is 6-base cutter (EcoRI). MseI 5’TTAA3’ EcoRI 5’GAATTC3’16
Adaptor Ligation - Two different adaptors (short double stranded DNA sequences with sticky end) are ligated to the digested fragments. - One adaptor will complement to the M cut sel end, the other will complement to the EcoRI cut end.17
AFLP20 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.
Electrophoresis - polyacrylamide gel is used for separating DNA bands. - Normally, 30-100 DNA bands can be detected by AFLP on polycrylamide gel.22
AFLP23 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 W hole genome analysis is (theoretically) possible The main disadvantage: complex procedure.
Polymorphism among 32 wheat samples revealed by AFLP25
Selective Bases - 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.26
Characteristics of AFLP - 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 large27
AFLP Data Map with UPGMA dendogram from Urbanelli et al. (2007): “Distinguishing taxa in the31 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