restriction enzyme digestion
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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 lengthc=concentration of substanceε=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/proteinA260/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 T4 L
(1.0% (w/v) agarose, EtBr staining)
L: Fermentas GeneRuler™ DNA Ladder Mix; 100-10000 bp DNA ladderZ: Zelha
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 methyltransferase-methylase) 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: HaeIII5’ 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
“sticky-ends”
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→GAATTCEcoR1 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
2,4kb
1kb0,6kb
6kb
1,5kb0,9kb0,8kb
0,6kb0,2kb
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
pBtSK+.seq2961 bps
500
1000
1500
2000
2500
FspIPvuII
KpnIApaIAvaIXhoIHincII
HindIIIEcoRVPstIAvaISmaIBamHI
Eco52IBstXISacI
PvuII
FspI
f1 ori
lacZT7
T3
lac promoter
ColE1
Ampicil l in
•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 DNAStocks typically at 10 u/ul