Restriction Enzymes

Dyah Ayu Oktavianie

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Restriction Enzymes & Ligase

These are used to make recombinant DNA. Two important first steps in cloning require two enzymes.

1. Restriction Enzymes Bacterial enzymes that cut at specific restriction site sequences Cut DNA by breaking phosphodiester bonds Yield restriction fragments that can be used to clone

2. DNA Ligase Enzyme reforms phosphodiester bond between adjacent nucleotides Page 2

Types of restriction enzymes

Type I Recognize specific sequencesbut then track along DNA (~1000-5000 bases) before cutting one of the strands and releasing a number of nucleotides (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the DNA
e.g. EcoK. Require Mg2+, ATP and SAM (S-adenosyl methionine) cofactors for function

Type II Recognize a specific target sequence in DNA, and then break the DNA (both strands), within or close to, the recognition site
e.g. EcoRI Usually require Mg2+

Type III Intermediate properties between type I and type II. Break both DNA strands at a defined distance from a recognition site
e.g. HgaI Require Mg2+ and ATP
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Restriction enzyme type II

Type II is the most frequent used in biology molecular techniques. Hundreds of restriction enzymes have been isolated and characterised Enables DNA to be cut into discrete, manageable fragments Many are now commercially available Each restriction enzyme will recognize its own particular site Some recognize more than one sequence Many Type II restriction endonucleases recognize PALINDROMIC sequences Restriction enzymes do not discriminate between DNA from different organisms
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The phosphodiester bond is cleaved between specific bases, one on each DNA strand

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5'-G A A T T C-3' 3'-C T T A A G-5'

Palindromic sequence

Generate 3' overhangs eg: PsfI

Generate 5' overhangs eg: EcoRI

Generate blunt end , eg: SmaI Page 6

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Examples of restriction enzymes and the sequences they cleave

Source microorganism
Arthrobacter luteus Bacillus amyloiquefaciens H Escherichia coli Haemophilus gallinarum Haemophilus infulenzae

Enzyme
Alu I Bam HI Eco RI Hga I Hind III

Recognition Site
AGCT GGATCC GAATTC GACGC(N)5 AAGCTT

Ends produced
Blunt Sticky Sticky Sticky Sticky

Providencia stuartii 164

Nocardia otitiscaviaruns Staphylococcus aureus 3A Serratia marcesans Thermus aquaticus

Pst I
Not I Sau 3A Sma I Taq I

CTGCAG
GCGGCCGC GATC CCCGGG TCGA

Sticky
Sticky Sticky Blunt Sticky Page 8

Ligation
When sticky ends are created on the vector and the rDNA, the ends are compatible and complementary Can be added as linkers or adapters DNA Ligase Seals single stranded nicks between adjacent nucleotides in a duplex DNA chain (catalyze the formation of a phosphodiester bond between adjacent 3 hydroxyl and 5 phosphate termini in DNA)

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widya-ugm

Constructing Genomic and cDNA Libraries

Definition

A cloned set of rDNA fragments representing either the entire genome of an organism (Genomic library) or the genes transcribed in a particular eukaryotic cell type (cDNA library) rDNA fragments generated using restriction endonucleases rDNA fragments ligated to appropriate cloning vector

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Genomic libraries

Commonly bacteriophage lambda used as the vector

Stuffer fragment removed and replaced with 15-17kbp fragments of library

Cosmids and YACs may also be used as vectors Contains at least one copy of all DNA fragments in the complete library Screened using nucleic acid probes to identify specific genes Subcloning is usually necessary for detailed analysis of genes
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CONSTRUCTION OF GENOMIC LIBRARY

GENE CLONING

1. Source of DNA 2. Enzyme : restriction endonucleases 3. Vector 4. Host

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SOURCE OF DNA
Isolated from the target organism (in which the genomics library will be directed) Decided the appropriate isolation method Prepared in high purity Fragmented using restriction endonuclease enzymes

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Common steps involved in isolating a particular DNA fragment from a complex mixture of DNA fragments or molecules
1.

2.

DNA molecules are digested with enzymes called restriction endonucleases which reduces the size of the fragments Renders them more manageable for cloning purposes These products of digestion are inserted into a DNA molecule called a vector Enables desired fragment to be replicated in cell culture to very high levels in a given cell

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3. Introduction of recombinant DNA molecule into

an appropriate host cell
Transformation or transfection Each cell receiving rDNA = CLONE May have thousands of copies of rDNA molecules/cell after DNA replication As host cell divides, rDNA partitioned into daughter cells

4. Population of cells of a given clone is expanded, and therefore so is the rDNA.

Amplification DNA can be extracted, purified and used for molecular analyses Investigate organization of genes Structure/function Activation Processing Gene product encoded by that rDNA can be characterized or widya-ugm modified through mutational experiments

RESTRICTION ENDONUCLEASE

Type I Type III Type II :*Palindromic :-blunt/flush end - sticky/cohesive end -3protruding
-5protruding

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ENDONUKLEASE RESTRIKSI

DASAR PEMILIHAN 1. Sekuen pengenalan : Kaitannya dengan DNA target . Kaitannya dengan tapak kloning pada vektor terpilih. 2. Panjang fragmen DNA yang diinginkan.
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