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.

•Restriction enzymes are molecular scissors – they cut DNA•restriction enzymes are highly specific. They cut DNA only within very precise recognition sequences.

• 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.

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

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.

• 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