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Recombinant DNA technology for protein production

1) Plasmid isolation using alkaline lysis method

2) Restriction digestion and Agarose gel electrophoresis

3) Transformation of Plasmid DNA

4) Protein isolation and SDS-PAGE


Restriction Digestion of Plasmid
DNA
Jeet Kalia
Basic structure of a Cloning and Expression Plasmid Vector

promoter

transcription
termination
Multiple cloning site sequence
(MCS)

Selection
marker
origin of
replication
Basic structure of a Cloning and Expression Plasmid Vector

MCS: This is a synthetic stretch of DNA and accomodates a string of unique


restriction sites. It facilitates cloning of a foreign DNA in an ORF w.r.t the promoter
Origin of replication (ori): This is a replicon of bacterial or phage origin and ensures
replication of plasmid DNA
Selection marker: This generally is a gene coding for an antibiotic resistance(
Ampicillin, Kanamycin, Tetracycline etc) and helps in selection of positive
transformants
Promoter: This generally is a viral promoter and is where the RNA polymerase
binds to drive the transcription of downstream foreign gene
Terminator sequence: This transcribes a stop codon downstream of the MCS
Pfu pET-17b: Plasmid Map

21-03-2017 BIO122 5
Source: Novagen
Restriction enzymes

Restriction enzymes/ restriction endonucleases (RE) :


a special class of endonucleases found in bacteria
digest DNA by recognizing specific short sequences of 4, 6
or 8 base pairs
Palindromic sequences
Palindromic sequence : that reads the same from both sides
Examples: 5-GAATTC-3
3-CTTAAG-5
Cleaves the phosphodiester bond of the DNA backbone
What are restriction enzymes?
Evolved by bacteria to protect
against viral DNA infection
1950s: discovery of primitive
immune system in bacteria
Werner Arber and Matthew
Meselson got the nobel
prize in 1960 for first
identifying these enzymes in
E. coli.
Host DNA is marked up by
Methylase enzyme- adds
methyl group to its bases
Endonucleases = cleave within DNA strands
Exonucleases = digest from the ends of DNA molecules
Types I, II, III and IV
3,139 known enzymes
Types of RE
Traditionally classified into four types on the
basis of :-
subunit composition
cleavage position
sequence specificity
cofactor requirements.
Type I REs
Complex, multisubunit and heterodimer,
combination restriction-and-modification enzymes
that cut DNA at random far from their recognition
sequences.
Cofactors: S-Adenosyl methionine (Ado-met),
hydrolyzed adenosine triphosphate (ATP), and Mg2+
ions.
Of considerable biochemical interest, but they have
little practical value since they do not produce
discrete restriction fragments or distinct gel-banding
patterns.
Type II REs
Homodimer, recognition sites are mostly palindromic (4-8 bp),
cleaves DNA near or within the recognition sites.
Do not use ATP; only requires Mg2+ ions as cofactors.
Subunits are in the range of 250-300 amino acids
Mostly recognize symmetric DNA sequences as they bind as
homodimers
The only class used in the laboratory for routine DNA analysis
and gene cloning since they produce discrete restriction
fragments and distinct gel banding patterns.
Palindromic Sequences
5 versus 3 overhang: Sticky Ends, subsequent
ligation is very specific
Enzyme cuts 5 GAATTC 3
3 CTTAAG 5

5 G 3
3 CTTAA 5
5 AATTC 3
3 G 5
Generates 5 overhang (in the 5 direction)
Blunt ends: subsequent ligation is non- 5 GAA TTC 3
specific 3 CTT AAG 5
Molecular cloning

Overhangs or sticky ends


Blunt ends
Type III REs
recognize two separate non-palindromic sequences
that are inversely oriented.
Cleave 20-30bp outside recognition sequences,
Contains more than one subunit
Requires ATP

Type IV REs
Recognizes modified, typically methylated DNA.
How are they named
Restriction enzymes are named by the
organism from which they were first isolated.
EcoRI Eco: E.coli R: I: first
strain enzyme of
that type
isolated
BamHI Bam: Bacillus H: I: first
amyloliquefaciens strain enzyme of
that type
isolated
Sau3A Sau: Staphylococcus 3A:
aureas strain
Restriction Enzyme Digestion
An Enzymatic Unit (u) is defined as the amount of enzyme required
to digest 1 ug of DNA under optimal conditions in 1 hour.

Restriction Buffer provides optimal conditions:


NaCl provides correct ionic strength
Tris-HCl provides the proper pH
Mg2+ is an enzyme co-factor

Why incubate at 370C?


Body temperature is optimal for these and most other enzymes.
What happens if temperature is too hot or cool?
How do we visualize the DNA?
Agarose gel electrophoresis
Agarose : polysaccharide extracted from the cell wall of red algae. The
three-dimensional matrix of the agarose gel is brought about by non-covalent
bonds formed between polysaccharide units.

Electrophoresis: a method of separating charged molecules in an electrical


field; DNA has an overall negative charge; it will move towards the positive
end (cathode to anode).

Size and Conformation (shape)


How do we visualize the DNA?
Cathode
-
Anode
+

Buffer
Dyes
Agarose
gel

Power Supply
Components of an Electrophoresis System
Electrophoresis Buffer:TAE (Tris-acetate-EDTA) and TBE (Tris-
borate-EDTA) are the most common buffers for duplex DNA.
Establish pH and provide ions to support conductivity.

Loading dye:DNA samples are loaded into a gel AFTER the tank
has been filled with buffer, covering the gel.
Contains a dense substance, such as glycerol,
to allow the sample to "fall" in the well

Contains one or two tracking dyes, which


migrate in the gel and allow monitoring of
how far the electrophoresis has proceeded.

DNA staining: Allow DNA visualization.


Ethidium bromide or Sybrsafe stains
Running plasmid DNA on an agarose gel

http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
Restriction Digestion Analysis
Protocol for restriction digestion
Component Volume
Sample Plasmid (DNA) 3 l
Restriction Enzyme 10X Buffer 2 l
Restriction Enzyme EcoRI 0.5 l
Restriction Enzyme HindIII 0.5 l
Water 14 l
Final volume 20l

Mix gently, close the tube and centrifuge for a few seconds.
Incubate at the 37 deg C (enzymes optimum temperature) for
45 min.
Protocol for Agarose Electrophoresis

1. Add loading buffer to a 1X final concentration and proceed to gel


electrophoresis.
2. Load the sample on 1% agarose gel and run it at 100 volts.
Examine the gel under the transilluminator once the dye front
traverses about 2/3 of the gel's length.
3. Capture the gel picture.
Questions: Restriction mapping
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