Arun Kumar. B M.Sc 1st Year Biotechnology SSBS RFLP, RAPD & AFLP Arun Kumar. B M.Sc 1st Year Biotechnology SSBS

RFLP RESTRICTION FRAGMENT LENGTH POLYMORPHISMS

Restriction Fragment Length Polymorphism RFLP analysis is a procedure used to show differences in the lengths of DNA fragments Different samples of DNA molecules have different lengths depending on where restriction enzymes cut them off Restriction enzymes are proteins that recognize specific sequences of DNA and cut the DNA at recognition sites, which vary between different samples of DNA, the location of the sites depends on the makeup of the particular DNA molecule This procedure of DNA analysis was widely used in the 1980’s, but became less and less popular as more efficient methods of DNA analysis were developed

PROCEDURE The DNA being analyzed has restriction enzymes added to it This DNA/enzyme mixture is left to sit, so the enzymes can cut at the recognition sites of the DNA molecule This “cut” DNA is run through a gel that separates DNA fragments according to their different sizes These DNA fragments are blotted onto a membrane and are analyzed.

USES This procedure has been used in genomic mapping and genetic disease analysis by seeing if certain family Members are at risk for genetically inherited diseases RFLP analysis can be used to see if someone is likely to be a carrier of mutant genes This procedure was used as an early basis for genetic fingerprinting, crime scene analysis, paternity tests, and characterization of genetic diversity in animal populations

ACCURACY & EFFICIENCY Although the procedure of RFLP Analysis is accurate, it also has drawbacks The entire process of RFLP analysis can take up to an entire month to complete A large amount of sample DNA must be given in order for the process to work, making it inefficient for use in crime scenes where only small traces of sample DNA can be collected Even though there are drawbacks of this procedure, it helped provide the foundation for the development of other procedures that are faster and more efficient.

How accurate is RFLP? RFLP paternity tests are 99% accurate. Prenatal testing for Huntington’s disease is 95% accurate Prenatal testing for Cystic Fibrosis is 99.5% accurate.

How is RFLP being used in today’s society? Allows scientists to map human genomes Helps to determine who is at risk of an inherited disease Genetic typing Genetic fingerprinting, profiling, or testing Provides genetic profile of a person Useful to compare samples at a crime scene Used to find a person’s paternity Used in forensics to solve crimes.

Paternity Testing

Disadvantages DNA contains over 1 million sites where a restriction enzyme will cut, but most restriction enzymes recognize DNA sequences that are only 6 bases long. The different size fragments cut from RFLP by restriction enzymes forms an unreadable continuous smear of DNA in an electrophoresis gel when accidently mixed. Requires large amounts of DNA. RFLP analysis is a slow process and can take up to a month to accomplish

How is it being misused? Patients identified as having a genetic disorder face genetic discrimination Insurance companies, employers, and other organizations have denied employment or insurance to individuals who are genetically prone to an illness. (The 2009 Genetic Information Nondiscrimination Act now makes this illegal.)

RAPD Randomly Amplified Polymorphic DNA

RAPD = random amplification of polymorphic DNA Principle: one short primer (e.g. 10 nucleotides) is used in the PCR reaction, this primer binds where it finds homology (many places), if two primers are by chance pointing to each other and not too far away, they can give a PCR product PCR-product primer DNA

How it works? In the RAPD technique, multiple 10 base pair (bp) oligonucleotide primers are added each to an individual sample of DNA which is then subjected to PCR. The resulting amplified DNA markers are random polymorphic segments with band sizes from 100 to 3000 bp depending upon the genomic DNA and the primer

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 nucleotides), and therefore, less specific. The primers can be designed without the experimenter having any genetic information for the organism being tested. More than 2000 different RAPD primers can be available commercially.

Genomic DNA normally has complimentary sequences to RAPD primers at many locations. If two of these locations are close to each other (<3000bp), and the sequences are in opposite orientation, the amplification will be established. This amplified region is said as a RAPD locus. Normally, a few (3-20) loci can be amplified by one single RAPD primer.

RAPD marker is a dominant marker RAPD marker is a dominant marker. Presence of a DNA band is dominant; absence of a DNA band is recessive. DNA bands of different sizes are assumed to be amplified products from different RAPD loci. RAPD Buffer (containing Mg++) - usually high Mg++ concentrations are used lowering annealing stringency Template DNA 1 short primer (10 bases)not known to anneal to any specific part of the template DNA dNTPs Taq DNA Polymerase (or another thermally stable DNA polymerase)

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

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

Primers are just the right distance apart, so fragment is amplified Template DNA 100 - 1,500 bases

Main advantages of RAPD: Simple technique (analysis on agarose gel) No sequence information needed to do this type of PCR Main disadvantage: Not very reproducible

AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS AFLP AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS

AFLP 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)

AFLP DNA First restriction digestion Second restriction digestion Adapter ligation

AFLP Selective fragment amplification Restriction enzyme digestion Adapter ligation common sequence 1 common sequence 2 Selective PCR amplification Selective bases AFLP primer 1 AC GC AFLP primer 2 Selective bases

Selected fragments are amplified (to have 50-150 bands on the gel) and separated by polyacrylamide gel electrophoresis (detection by 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.

Pros & Cons 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

REFERENCES http://www.wiley.com/college/boyer/0470003790/cutting_edge/dna_fingerprinting/DNAFingr.htm

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