BITO-111BIOTECHNOLOGY

AYESHA MASRUR KHANDECEMBER 2013

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More on Restriction Enzymes

Restriction enzymes are Nucleases which can cleave the sugar-phosphate backbone of DNA, found in bacteria. As they cut within the molecule, they are commonly called restriction endonucleases. They specifically cleave the nucleic acids at specific nucleotide sequence called Restriction sites to generate a set of smaller fragments .

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More on Restriction Enzymes• Different restriction enzymes can have the same

recognition site (known as ‘isoschizomers’)– Different recognition sites can also produce identical

overhangs (known as compatible ends)• Recognition sites can be ambiguous or unambiguous,

e.g. HinfI recognizes 5 bp sequence starting with GA, ending in TC with any base in between, or XhoII recognizes AGATCT, AGATCC, GGATCT and GGATCC.

• The recognition site for one enzyme may contain the restriction site for another.

• Most recognition sites are palindromes, but not all.

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And more…

Possibility of the restriction fragments to stick back together in the plasmid

How is it prevented?1. Treat the plasmid with phosphatase to prevent re-ligation. -removal of 5’ phosphate group from the plasmid DNA 2. Use two different restriction enzymes to cut the plasmid DNA and donor DNA. -generation of incompatible sticky ends on the plasmid

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2 possibilities:

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Reaction conditions and requirements for Restriction enzymes

pH, ionic strength & temperature are specific for each restriction enzymeIncubation time depends on DNA concentration & amount of the enzymeDigestion can be carried out using mixtures of restriction enzymes Magnesium (Mg2+) requirement, or any other divalent cation

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Action of methylase

Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own enzymes

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Alkaline phosphatases

Two primary uses for alkaline phosphatases in DNA manipulation:

Removal of 5’ phosphates from plasmid and bacteriophage vectors that have been cut with a RE.

-In subsequent ligation reactions, this treatment prevents self-ligation of the vector, making ligation of other DNA fragments feasible.

Removing 5’ phosphates from fragments of DNA prior to labeling with radioactive phosphate.

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Other important enzymesDNA polymerase:Synthesis of DNA complementary to a DNA template in the 5’ to 3’ direction.

Exonuclease:Trimming of 3’ overhangs to make them blunt ended.

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Terms to shed light on:Transformation: Uptake of exogenous genetic

material by bacterial cell.Transfection: Uptake of exogenous genetic

material, through non viral methods, in eukaryotic cells.

Transduction: Also a form of transformation but refers specifically to uptake of phage DNA by host.

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Restriction DigestWhat is a restriction digest ?Pattern of cutting/splicing of an entire length of a DNA molecule

What does it tell us?Difference between related DNA molecules, by separating the various restriction fragments produced by various restriction enzymes

How is it carried out?1. Electrophoresis –Gels used are Polyacrylamide gel, Agarose

gel2. Appropriate Staining-Ethidium bromide3. Visualize and compare

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What can affect generation of a restriction digest

Changes in the gene sequence that are found in introns or between genes>>>polymorphismRestriction enzymes can generate variable sizes of fragments due to this polymorphism >>>RFLP (restriction fragment length polymorphism)

e.g. 1. If two restriction sites are present and one of them has a single base

altered, then the restriction enzyme will produce a single large fragment instead of two fragments.

2. Insertion or deletion of sequences between two splice sites means that fragments size may increase or decrease.

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Restriction MappingDescription of restriction endonuclease cleavage sites within a piece of DNA.

It is useful for:Characterizing an unknown DNAPre-requisite for manipulation

3000bp of unknown DNA in plasmid

KpnI

BamHI

1000bp+’big’ (2000bp + vector DNA)

600, 2200 and ‘big’ (200 bp + vector DNA)

Digest plasmid with KpnI & BamHI together (double digest)

600, 1000, 1200 bp + ‘big’

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Note: KpnI & BamHI flank the unknown DNA

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Analysis of DNA molecules

Gel ElectrophoresisWe can differentiate DNA molecules based on two properties ---DNA fragment size and restriction sites

Electrophoresis separates molecules based on their size and/or electrical charge

•Medium containing agarose consists of an interlocked fiber matrix.•Phosphate groups give DNA a very strong negative charge. •The DNA samples (different sized fragments) are loaded into wells at one end of the prepared agarose gel.•The DNA molecules will migrate to the opposite end when electric current is applied.

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Visualization

•Stain the DNA with a fluorescent dye to make it visible.

•After completion of electrophoresis soak the gel in a solution of ethidium bromide (EtBR) or add it in the gel while the gel is being prepared. Alternately it can also be added to the samples of DNA.

-it binds tightly to the DNA molecules and is flourescent (bright orange color) under UV light.

•Run the samples side by side with DNA molecules of known length (size standards).

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Factors that affect migration

>Agarose concentrationHigher concentrations of agarose facilitates separation of small DNAs; while low agarose concentrations allow resolution of larger DNAs.

>Shape of the nucleic acidsSupercoiled circular DNA, relaxed circular DNA & linear DNA of the same molecular weight will migrate at different rates through the gel.

>Voltage appliedAt higher voltage, larger fragments migrate proportionally faster than small fragments.

>Electrophoresis bufferDNA fragments migrate at different rates in different buffers due to differences in ionic strengths & pH.

>Ethidium bromideBinding affects the mobility of DNA