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RFLP
RESTRICTION FRAGMENT LENGTH POLYMORPHISMS
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
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.
Genetic typing
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.
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
primer
DNA
PCRproduct
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.
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
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
Templat e DNA
Only when primer binding sites are close and oriented in opposite direction so the primers point toward each other will amplification take place
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
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
Adapter ligation
AFLP
Adapter ligation
common sequence 1 common sequence 2
GC
AFLP primer 2
Selective bases
27
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.
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
REFERENCES
http://www.wiley.com/college/boyer/0470003790/cutting_edge/d na_fingerprinting/DNAFingr.htm