Restriction Enzymes

• Restriction Enzymes scan the DNA sequence• Find a very specific set of nucleotides• Make a specific cut

Palindromes in DNA sequences

Genetic palindromes are similar to verbal palindromes. A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands.

5’

5’

3’

3’

Restriction enzymes recognize and make a cut within specific

palindromic sequences, known as restriction sites, in the DNA. This is

usually a 4- or 6 base pair sequence.

Restriction Endonuclease Types

Type I- multi-subunit, both endonuclease and methylase activities, cleave at random up to 1000 bp from recognition sequence

Type II- most single subunit, cleave DNA within recognition sequence

Type III- multi-subunit, endonuclease and methylase about 25 bp from recognition sequence

Hae IIIHaeIII is a restriction enzyme that

searches the DNA molecule until it finds this sequence of four nitrogen bases.

5’ TGACGGGTTCGAGGCCAG 3’3’ ACTGCCCAAGGTCCGGTC 5’

Once the recognition site is found Hae III will cleave the DNA at that site

5’ TGACGGGTTCGAGGCCAG 3’3’ ACTGCCCAAGGTCCGGTC 5’

These cuts produce “blunt ends”

5’ TGACGGGTTCGAGG CCAG 3’3’ ACTGCCCAAGGTCC GGTC 5’

The names for restriction enzymes come from:

• the type of bacteria in which the enzyme is found• the order in which the restriction enzyme was identified

and isolated.

EcoRI for exampleR strain of E.coli bacteria

I as it is was the first E. coli restriction enzyme to be discovered.

“blunt ends” and “sticky ends”Hae III produced a “blunt end”?

EcoRI makes a staggered cut and produces a “sticky end”

5’ GAATTC 3’3’ CTTAAG 5’

5’ GAATTC 3’3’ CTTAAG 5’

5’ G AATTC 3’3’ CTTAA G 5’

blunt end

sticky end

More examples of restriction sites of restriction enzymes with their cut sites

Hind III: 5’ AAGCTT 3’ 3’ TTCGAA 5’

Bam HI: 5’ GGATCC 3’ 3’ CCTAGG 5’

Alu I: 5’ AGCT 3’ 3’ TCGA 5’

Separating Restriction Fragments, I

Separating Restriction Fragments, II

Gene Cloning

• What is gene cloning? How does it differ from cloning an entire organism?

• Why is gene cloning done?• How is gene cloning

accomplished ?• What is a DNA ‘Library’?

What is DNA cloning?

• When DNA is extracted from an organism, all its genes are obtained

• In gene (DNA) cloning a particular gene is copied (cloned)

Why Clone DNA?• A particular gene can be isolated and its

nucleotide sequence determined• Control sequences of DNA can be

identified & analyzed• Protein/enzyme/RNA function can be

investigated• Mutations can be identified, e.g. gene

defects related to specific diseases• Organisms can be ‘engineered’ for

specific purposes, e.g. insulin production, insect resistance, etc.

How is DNA cloned?, I

• DNA is extracted- here from blood

• Restriction enzymes, e.g. EcoR I, Hind III, etc., cut the DNA into small pieces

• Different DNA pieces cut with the same enzyme can join, or recombine.

Blood sample

DNA

Restriction enzymes

The action of a restriction enzyme, EcoR INote: EcoR I gives a ‘sticky’ end

DNA Cloning, II

• Bacterial plasmids (small circular DNA additional to a bacteria’s regular DNA) are cut with the same restriction enzyme

• A chunk of DNA can thus be inserted into the plasmid DNA to form a “recombinant”

DNA cloning, III

• The recombinant plasmids are then mixed with bacteria which have been treated to make them “competent”, or capable of taking in the plasmids

• This insertion is called transformation

DNA Cloning, IV

• The plasmids have naturally occurring genes for antibiotic resistance

• Bacteria containing plasmids with these genes will grow on a medium containing the antibiotic- the others die, so only transformed bacteria survive

DNA Cloning, V

• The transformed bacterial cells form colonies on the medium

• Each cell in a given colony has the same plasmid (& the same DNA)

• Cells in different colonies have different plasmids (& different DNA fragments)

Screening, IScreening can involve:1. Phenotypic

screening- the protein encoded by the gene changes the color of the colony

2. Using antibodies that recognize the protein produced by a particular gene

Screening, II 3. Detecting the DNA sequence of a

cloned gene with a probe (DNA hybridization)

Polymerase Chain Reaction

PCR

PCR

• invented by Karry B. Mullis (1983, Nobel Prize 1993)

• patent sold by Cetus corp. to La Roche for $300 million

• depends on thermo-resistant DNA polymerase (e.g. Taq polymerase) and a thermal cycler

Heat-stable DNA polymerase

• Taq DNA polymerase was isolated from the bacterium Thermus aquaticus.

• Taq polymerase is stable at the high temperatures (~95oC) used for denaturing DNA.

Hot springs at Yellowstone National Park, Wyoming.

DNA polymerase requirements

• template• primer• nucleotides• regulated pH, salt concentration,

cofactors

Steps in DNA replication

1) template denatured

2) primers anneal

3) new strand elongation

Steps in a PCR cycle1) template denatured:

94 C, 30 sec 2) primers anneal

45-72 C, depending on primer sequence30 sec – 1 min

3) new strand elongation72 C depending on the type of polymerase1 min for 1000 nucleotides of amplified sequence

Number of specific DNA molecule copies grows exponentially with each PCR cycle. Usually run 20-40 cycles to get enough DNA for most applications (If you start with 2 molecules, after 30 cycles you will have more than a billion)

PCR Process• 25-30 cycles

• 2 minute cycles

• DNA thermal cycler

Annealing primers

New strand elongation

Template denatured

Uses for PCR

• Research– Gene cloning

– Real-time PCR

– DNA sequencing

• Clinical– DNA fingerprinting

• Crime scene analysis• Paternity testing• Archeological finds

– Genetically inherited diseases

DNA Sequencing

Chain termination method (Sanger Method), sequence of single stranded DNA is determined by enzymatic synthesis of complementary strands which terminate at specific nucleotide positions

Chemical degradation method (Maxam-Gilbert Method), sequence of a double stranded DNA molecule is determined by chemical treatment that cuts at specific nucleotide positions

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.604

Dideoxynucleotide (ddNTP)

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http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.6477

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.607

Costs and time for sequencing a human genome (3.2 billion bp)