AYESHA MASRUR KHAN DECEMBER 2013

More on Restriction Enzymes 2 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.

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. 3

And more… 4 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

5 2 possibilities:

Reaction conditions and requirements for Restriction enzymes  pH, ionic strength & temperature are specific for each restriction enzyme  Incubation time depends on DNA concentration & amount of the enzyme  Digestion can be carried out using mixtures of restriction enzymes  Magnesium (Mg2+) requirement, or any other divalent cation  pH, ionic strength & temperature are specific for each restriction enzyme  Incubation time depends on DNA concentration & amount of the enzyme  Digestion can be carried out using mixtures of restriction enzymes  Magnesium (Mg2+) requirement, or any other divalent cation 6

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 7

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. 8

Other important enzymes DNA polymerase: Synthesis of DNA complementary to a DNA template in the 5’ to 3’ direction. 9 Exonuclease: Trimming of 3’ overhangs to make them blunt ended.

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. 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. 10

Restriction Digest BITO What 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 gel 2.Appropriate Staining-Ethidium bromide 3.Visualize and compare

What can affect generation of a restriction digest BITO Changes in the gene sequence that are found in introns or between genes>>>polymorphism Restriction 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.

Restriction Mapping BITO Description of restriction endonuclease cleavage sites within a piece of DNA. It is useful for:  Characterizing an unknown DNA  Pre-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’ Note: KpnI & BamHI flank the unknown DNA

Analysis of DNA molecules BITO Gel Electrophoresis We 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.

Visualization BITO 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).

Factors that affect migration BITO >Agarose concentration Higher concentrations of agarose facilitates separation of small DNAs; while low agarose concentrations allow resolution of larger DNAs. >Shape of the nucleic acids Supercoiled circular DNA, relaxed circular DNA & linear DNA of the same molecular weight will migrate at different rates through the gel. >Voltage applied At higher voltage, larger fragments migrate proportionally faster than small fragments. >Electrophoresis buffer DNA fragments migrate at different rates in different buffers due to differences in ionic strengths & pH. >Ethidium bromide Binding affects the mobility of DNA