restriction enzymes/endonucleases restriction enzymes are proteins that scan the dna for specific...
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Restriction Enzymes/endonucleases
Restriction enzymes are proteins that scan the DNA for specific sequences, usually palindromic sequences of 4 to 8 nucleotides, and then cleave both strands at that position.
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•Restriction enzymes are molecular scissors – they cut DNA•restriction enzymes are highly specific. They cut DNA only within very precise recognition sequences.
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• There are many different restriction enzymes that can be used.
• To detect variation in DNA sequence restriction enzyme digestion can be used.
• Variation in the DNA sequence that gives rise to the creation or destruction of a restriction enzyme digestion site is called a restriction fragment length polymorphism (RFLP).
• Humans have two copies of each gene, except for those genes on the X and Y chromosomes in males. This must be kept in mind when doing RFLP analysis. In each sample there may be two alleles.
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Examples of Restriction Enzymes
Enzyme Organism from which derived Target sequence(cut at *)5' -->3'
Ava I Anabaena variabilis C* C/T C G A/G G
Bam HI Bacillus amyloliquefaciens G* G A T C C
Bgl II Bacillus globigii A* G A T C T
Eco RI Escherichia coli RY 13 G* A A T T C
Eco RII Escherichia coli R245 * C C A/T G G
Hae III Haemophilus aegyptius G G * C C
Hha I Haemophilus haemolyticus G C G * C
Hind III Haemophilus inflenzae Rd A* A G C T T
Hpa I Haemophilus parainflenzae G T T * A A C
Kpn I Klebsiella pneumoniae G G T A C * C
Sma I Serratia marcescens C C C * G G G
Sal I Streptomyces albus G G * T C G A C
Xma I Xanthamonas malvacearum C * C C G G G
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The original procedure used to obtain a DNA fingerprint
1. Isolate genomic DNA
2. cut with restriction enzymes
3. run on a gel• As humans have more than 3 billion base pairs in their genome , after
electrophoresis all that can be seen is a smear because all the resulting bands overlap.
• To visualise a fingerprint pattern the DNA fragments must be detected by probing and hybridisation.
4. After electrophoresis the DNA in the gel is denatured and transferred to a membrane to make a permanent record.
5. The membrane is then “probed” using a piece of sequence that is complimentary to a hypervariable region.
6. The binding of the probe is visualised using radioactivity, fluorescence, conjugated enzyme.
7. The resulting band patterns are a fingerprint.
8. The final DNA fingerprint is built by using several probes (5-10 or more) simultaneously.
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• Today because we have the human DNA sequence and certain other genome sequences instead of digesting total genomic DNA and creating a permanent record on a membrane that is then probed for variable regions , several different highly variable regions are amplified directly by PCR
• FBI uses 22 different regions, RCMP 15 different regions, paternity tests typically use at least 7 different regions
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