RFLP: Restriction Fragment Length Polymorphism • A restriction enzyme cuts the DNA molecules at every occurrence of a particular sequence, called restriction site. For example, HindII enzyme cuts at GTGCAC or GTTAAC • Any mutation of a single nucleotide may destroy or create the site (AACCTT instead of AAGCTT for Hind III) • RFLP analysis is the detection of the change in the length of the restriction fragments. RFLPs associated with base changes that alter restriction endonuclease sites. After the cleavage we will obtain DNA fragments of different molecular weight Restriction endonucleases cut DNA molecules at defined positions If a mutation occurs in X chromosome in a restriction site an individual may appear as • Normal GAATTC • Mutant GAGTTC The internal EcoRI site is missing in the second individual • sequence recognized by EcoRI GAATTC CTTAAG • Mutated Sequence not recognized by the enzyme GAGTTC CTCAAG Sickle Cell anemia with RFLP • Sickle Cell anemia is caused by a mutation in the beta-globin gene. • The difference between the normal BA allele and the sickle-cell BS allele is a single-nucleotide substitution (A to T) . • The sequence of the standard BA allele (CCTGAGG) is cut with MstII restriction enzyme .This sequence is altered in the BS allele (CCTGTGG). • In the genetic test for the BS allele, total DNA from the individual tested is digested with MstII and run in a southern blot. • If the normal BA allele is present, the probe produces two smaller bands. • If the sickle-cell BS allele is present, the probe produces one larger band. • Thus, a standard AA homozygote shows the two-band pattern, a SS homozygote (with sickle-cell anemia) shows the one-band pattern, and an AS heterozygote (with sickle-cell trait) shows all three bands. normal BA = two bands sickle-cell BS allele = one larger band AS heterozygote = all three bands Gel-Electrophoresis • DNA is cut into fragments using an enzyme • The cut DNA is put on a Gel material • An electric current is applied on the Gel • DNA fragments will start moving towards the positively charged side . Smaller fragments move faster. • After some time, we have a separation of the different fragment lengths Restriction Enzyme • A restriction enzyme is used to cut the DNA into fragments • Hind III restriction site is A AGCTT Apply Enzyme DNA sample and Hind III are put together in a tube • The tube is shaken by rotation for DNA and Hind III to mix Water Bath The tube is put on a plate floating on water at 37oC • It is left for 30 minutes. • This is needed for the Hind III reaction to take place Preparing the Gel The liquid Gel is poured into the inner box • A comb like piece is put at the edge of the inner box • The liquid Gel is left to cool and solidify . • When the Gel solidifies, the comb will create wells for the DNA samples to be put Loading DNA on the Gel DNA samples mixed with colored solution and UV reactive solution • DNA samples inserted into wells • A sample DNA containing size marker for comparison Run the Gel •Apply electric current • DNA is negatively charged • Fragments will migrate toward the positive charge • Small fragments move faster Viewing • Original uncut DNA sample makes a sharp band at the beginning (one big fragment) • DNA sample cut with Hind III makes smear (lots of fragments of all sizes) • Ladders are used for comparison. • We could run it for a longer time to achieve better separation Use of restriction enzymes in forensics – DNA fingerprinting • DNA is subjected to restriction enzyme, each individual will generate different sizes of DNA fragments . • The restricted DNA fragments are then exposed to gel electrophoresis • Gel electrophoresis separates the DNA fragments depending on the size.