Download Molecular Biology-restrection enzyme

Molecular Biology
March 2007
Restriction Enzymes
Restriction Enzymes
• Restriction enzymes are part of the defense system of
bacteria: they digest foreign DNA that enters the bacterial
cell.
• Each species of bacteria has its own set of restriction
enzymes. Each enzyme cuts DNA at a specific short
base sequence. For instance, EcoR1 cuts the DNA at the
sequence GAATTC, and BamH1 cuts at GGATCC. There
are hundreds of restriction enzymes known.
• Using properly chosen enzymes, the gene you want can be
cut out of the chromosome intact, with very little extra DNA.
• Many restriction enzymes give a staggered cut across
the DNA double helix. This produces short single stranded
regions, called “sticky ends”. The ends are sticky because
they spontaneously pair with similar ends.
Restriction Endonucleases
z Restriction endonucleases cut DNA
z Present in bacteria
z Cut at sequence specific sites
– Usually 4 or 6 base pairs long
z Bacteria use them to destroy ‘foreign’ DNA
– Bacteria protect their own DNA against self-cutting
by special methylation of their DNA
z Restriction enzymes can be purified and are
used in genetic engineering studies
Restriction Endonucleases
• Example Restriction enzymes
– EcoR I (E. coli Restriction Endonuclease I)
– Stu I (Streptomyces tubercidicus I)
EcoR I
Palindromic
Axis of rotational symmetry
5’
3’
GAATTC
3’
5’
CTTAAG
Sticky Ended
Stu I
5’
3’
AGGCCT
3’
5’
TCCGGA
Blunt Ended
Molecular Scissors and Glue
zThere are 200’s of restriction
enzymes, each one with a
different recognition site
– These enzymes are ‘molecular
scissors’ and can be used to
specifically cut long DNA strands
into smaller pieces
Restriction Enzymes
¾ called "restriction enzymes“ because restrict host range for certain
bacteriophage
¾ bacterial" immune system": destroy any "non-self" DNA
¾ methylase recognizes same sequence in host DNA and protects it by
methylating it; restriction enzyme destroys unprotected = non-self DNA
(restriction/modification systems)
Restriction Enzymes
¾ Most recognize and cut
palindromic sequences
¾ Many leave staggered (sticky)
ends
¾ by choosing correct enzymes
can cut DNA very precisely
¾ Important for molecular biologists
because restriction enzymes
create unpaired "sticky ends"
which anneal with any
complementary sequence
¾ Hundreds of restriction enzymes
have been identified.
Some Commonly Used
Restriction Enzymes
Eco RI 5'-G | AATTC
Eco RV 5'-GAT | ATC
Hin D III 5'-A | AGCTT
Sac I 5'-GAGCT | C
Sma I 5'-CCC | GGG
Xma I 5'-C | CCGGG
Bam HI I 5'-G | GATCC
Pst I I 5'-CTGCA | G
Theoretical Basis
Using Restriction Enzymes
¾
The activity of restriction enzymes is dependent
upon precise environmental condtions:
PH
Temperature
Salt Concentration
Ions
¾
An Enzymatic Unit (u) is defined as the amount of
enzyme required to digest 1 ug of DNA under optimal
conditions:
3-5 u/ug of genomic DNA
1 u/ug of plasmid DNA
Stocks typically at 10 u/ul
Agarose gel electrophoresis of the PCR products
for the 3'UTR of the leptin receptor gene. Lane 1: a
100 bp ladder, lanes from 2 to 20: the amplified
fragments of the 3'UTR region.
A PCR assay for the CTTTA insertion at the
3'UTR of the leptin receptor gene
A PCR assay for the CTTTA insertion at the 3'UTR of the leptin receptor
gene. A fragment of 114 bp or 119 bp in size was amplified, digested with
the enzyme Rsal and analyzed by agarose gel electrophoresis, Lane 1:
molecular size marker (a 100 bp ladder); lane 2,7and 10: undigested
sample, lanes 3, 4, 5, 6 and 9: individuals with genotypes heterozygous(
del /ins, - + ), lanes 6 and 8: individuals with genotypes homozygous (
ins/ins, + + ).
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