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