Download Restriction Enzyme Digestion

Restriction Enzyme Digestion
Zelha Nil
Nov-09
Today’s Laboratory Objectives
• Results of gDNA experiment: the concentration,
purity, and integrity of genomic DNA
• Digest genomic DNA and plasmids
DNA quantification
• A UV spectophotometer measures the amount of light
particular molecules absorb (Proteins at A280; Nucleic
Acids at A260)
• Lambert-Beer law describes the relationship between
absorptivity coefficient and concentration and is given by
the following equation:
A=εbc
Where:
b= light path length
c=concentration of substance
ε=extinction coefficient
For DNA the extinction coefficient, ε= 50 ug/ml
DNA quantification
• To Quantify your DNA sample:
A260 x Dilution Factor x 50 ug/ml= concentration of
nucleic acids in a sample using a 1 cm pathlength
(DF=200)
• To estimate the purity of your sample:
A260/A280= ratio of nucleic acids/protein
A260/A280= 1.6-1.8 is optimal for DNA
Integrity of genomic DNA
• High Quality Genomic DNA
>95% DNA will be of high molecular
weight, migrating as intact band
near the top of the gel
Very little evidence of smaller
fragments indicated by a smear of
many different sized DNA fragments
Our results
L
Z
W1
W2
W3
W4
L
Z
T1
T2
T3
(1.0% (w/v) agarose, EtBr staining)
L: Fermentas GeneRuler™ DNA Ladder Mix; 100-10000 bp DNA ladder
Z: Zelha
T4
L
Restriction Enzymes
• Phage (or viruses) invade all types of cells. Bacteria are
one favorite target.
• Defense mechanisms have been developed by bacteria
to defend themselves from these invasions.
• Bacteria have evolved a class of enzymes that destroy
foreign DNA (eg. Virus DNA).
▫ protect bacteria from bacteriophages (Viruses).
• Infecting DNA is cleaved (restricted) by the restriction
enzyme(s) preventing it from successfully replicating and
parasitizing the cell.
Why the bacteria does not kill itself?
The Restriction Enzyme Modification Systems
If everything gets cleaved, how come the bacteria does not kill
itself?
• Usually, organisms that make restriction enzymes also make a
companion modification enzyme (DNA methyltransferasemethylase) that protects their own DNA from cleavage.
• These enzymes recognize the same DNA sequence as the
restriction enzyme they accompany, but instead of cleaving
the sequence, they disguise it by methylating one of the
bases in each DNA strand.
RE system
• This system is composed of a restriction
endonuclease enzyme and a methylase enzyme
• Each bacterial species and strain has their own
combination of restriction and methylating enzymes.
• Restriction endonuclease is an enzyme that cuts DNA
at internal phosphodiester bonds; different types
exist and the most useful ones for molecular biology
are those which cleave at a specific DNA sequence.
Classification of restriction enzymes
• Type 1:
▫ One enzyme with different subunits for recognition,
cleavage, & methylation.
▫ The methylation and cutting rxns both require ATP, Mg+2
and S-adenosylmethionine as cofactors.
▫ The enzyme cuts unmodified DNA at some distance (~1000
bp away) from the recognition site (Asymmetrical
recognition sequences).
• Type 2s:
▫ Asymmetric recognition sequence & cleavage occurs on one
side of recognition sequence up to 20 bp away.
• Type 3:
▫ Resemble type 1 systems but have symmetrical recognition
sequences.
• Type 2:
▫ Restriction and modification are mediated by
separate enzymes so it is possible to cleave DNA in
the absence of modification.
▫ The restriction activities do not require cofactors,
making them easier to use.
▫ Most importantly; those enzymes recognize a
defined, usually symmetrical sequence and cut
within it.
Nomenclature
• Smith and Nathans (1973) proposed enzyme
naming scheme;
▫ Three-letter acronym for each enzyme derived from
the source organism
▫ First letter from genus
▫ Next two letters represent species
▫ Additional letter or number represent the strain or
serotypes
• For example. the enzyme HindII was isolated
from Haemophilus influenzae serotype d.
• Most type 2 RE recognize and cleave DNA within
particular sequences of 4 to 8 nucleotides which
have two fold axis of rotational symmetry. Such
sequences are often referred as palindromes:
• Ex: HaeIII
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Ends of restriction fragments;
• Blunt ends
• Sticky ends
▫ 3‘ extensions
▫ 5‘ extensions
• Importantly, the 5' termini of each strand in the
cleavage product(s) retain the phosphoryl group
from the phosphodiester bond, the 3' termini
are hydroxylated.
Blunt ends
• Some restriction enzymes cut DNA at opposite
base
• They leave blunt ended DNA fragments
AluI
HaeIII
Sticky ends
• Most restriction enzymes make staggered cuts
• Staggered cuts produce single stranded “stickyends”
Star effect
• Optimum conditions are necessary for the
expected result.
• Under extreme conditions such as elevated pH
or low ionic strength, RE are capable of cleaving
sequences which are similar but not identical to
their recognition sequence.
• EcoR1→GAATTC
EcoR1 with star activity→NAATTN
(N=any base)
General uses of REs
• Detection of RFLPs
• Restriction enzyme map: The location of the
restriction enzyme cleavage sites on the DNA
molecule
• DNA fragments from different species can be
ligated to create Recombinant DNA:
Example
Single digest with EcoRI:
Double digest with EcoRI & PstI:
6kb
6kb
2,4kb
1,5kb
1kb
0,6kb
0,9kb
0,8kb
0,6kb
0,2kb
Experimental procedure
• Genomic DNA isolated last week and the
plasmid DNA isolated before will be digested.
• Single digestion with EcoRI
• Double digestion with EcoRI & HindIII
f1 ori
Ampicillin
FspI
2500
FspI
PvuII
KpnI
ApaI
AvaI
XhoI
HincII
500
pBtSK+.seq
T7lacZ
T3
2961 bps
Eco52I
BstXI
SacI
lac promoter
2000
1000
1500
PvuII
ColE1
HindIII
EcoRV
PstI
AvaI
SmaI
BamHI
• Group 1 &3: Single digestion
• Group 2&4: Double digestion
• An Enzymatic Unit (u) is defined as the amount of
enzyme required to digest 1 ug of DNA under optimal
conditions:
2-3 u/ug of genomic DNA
1 u/ug of plasmid DNA
Stocks typically at 10 u/ul