CLONING DNA PART II

REVIEW: CHALLENGE

REMEMBER THIS?

GENE CLONING Three steps: 1.Creation of recombinant DNA 2.Bacterial transformation 3.Screening for successful clones

BACTERIAL TRANSFORMATION Two main methods to help bacteria pick up plasmid: a)Cold CaCl 2 treatment followed by heat-shocking makes cell membrane leaky and more permeable b)Electroporation electric current to increase cell permeability

QUESTION How will you know which bacteria pick up the correct vector? Remember: plasmids contain a gene for antibiotic resistance

PLATES Bacteria are grown on Petri plates containing agar. Nutrients are mixed within agar providing a food source.

SCREENING FOR SUCCESSFUL CLONES DETERMINING IF AND WHICH BACTERIA TOOK UP THE PLASMID VECTOR Add antibiotics to the agar If bacteria grow: contain antibiotic-resistant vector If bacteria don’t grow: don’t contain antibiotic- resistant vector

SCREENING FOR SUCCESSFUL CLONES A Some inserts are designed to interrupt genes on the vector such as the Lac Z gene LacZ gene codes for β–galactosidase activity X-gal added to agar (X gal is an analogue of lactose and can be broken down by β–galactosidase ) X- gal itself is colourless The breakdown products of X-gal are blue (this indicates active LacZ gene;therefore no transformation)

SCREENING Vector is designed so that an insert will disrupt lacZ gene – what does this mean? Clones plated on agar plates containing both antibiotic and X-gal

SCREENING

Successful clones are white-coloured colonies This technique of using X-gal as a visual indication of whether a gene was successfully inserted so as to interrupt the LacZ gene is called blue/white screening. BLUE = UNSUCCESSFUL (NO GENE CLONING in that bacteria, β–galactosidase is active) This method of screening is a convenient way of distinguishing a successful cloning product from other unsuccessful ones. Clones are transferred to larger containers to be grown in greater concentrations LacZ is used as a reporter gene since it indicates

HYBRIDIZATION PROBES Hybridization Probe: fragment of DNA used to detect presence of complementary nucleotide sequences Can be used to detect presence of gene in plasmid

POLYMERASE CHAIN REACTION (PCR)

PCR PCR: a synthetic method (no living cells) to produce millions of copies of DNA use enzymes in a tube - in vitro The more DNA available, the easier it is to work with. Minimum requirements for DNA polymerase: 1.Template strand 2.Primers 3.Nucleoside triphosphates

STEPS OF PCR 1.DNA strand denaturation(95°C) separate double stranded DNA each strand becomes template strand 2.Primer annealing(50°C - 65°C) bind short DNA pieces (primers) to template strands 3.DNA strand synthesis(72°C) produce new DNA strands

PROBLEM Where do you find enzymes that don’t break down at 95°C? Thermus aquaticus bacterium live in hot springs and their enzymes are designed to withstand extreme temperatures. Isolated Taq polymerase from these bacteria.

PCR

After 30 cycles, 2 30 (more than a billion) copies of DNA can be produced. 30 cycles of PCR can take anywhere from 1 – 2 hours to complete.

PCR APPLICATIONS Genetic Screening Early detection for certain diseases. Forensic Analysis Small amounts of DNA collected to identify individuals. Gene Cloning Create many copies of the gene to be inserted into the plasmid

GEL ELECTROPHORESIS

Gel electrophoresis: moving DNA through a gel medium using an electric current Liquid solutions of the gel is poured into a mould and allowed to set and solidify. Why can we move DNA with electricity? DNA has a negative charge.

GEL ELECTROPHORESIS

Each well is loaded with a different sample of DNA. Samples often contain multiple pieces of DNA of different lengths.

GEL ELECTROPHORESIS The gel provides resistance for DNA movement. Short DNA moves through gel easily travels further in a set amount of time Long DNA requires more effort to move through gel does not move as far in a set amount of time

GEL ELECTROPHORESIS The gel medium can be made from: 1. agarose: seaweed extract 2. polyacrylamide: artificial polymer The type of gel used is dependent on how well separated the DNA pieces need to be (resolution). Polyacrylamide has higher resolution than agarose.

GEL ELECTROPHORESIS DNA is colourless. How do you know that it gets into the gel? Coloured dyes are mixed with DNA to track distance travelled. How do you see where the DNA is after the separation is complete? DNA is stained with ethidium bromide and a UV light box is used to see fluorescent DNA bands

GEL ELECTROPHORESIS DNA is colourless. How do you know that it gets into the gel? Coloured dyes are mixed with DNA to track distance travelled.

GEL ELECTROPHORESIS How do you see where the DNA is after the separation is complete? DNA is stained with ethidium bromide and a UV light box is used to see fluorescent DNA bands

GEL ELECTROPHORESIS How do you know how long your DNA is?

GEL ELECTROPHORESIS A DNA ladder is also loaded on the gel containing known lengths of DNA. Samples of unknown length can then be approximated through comparison with the DNA ladder. The ladder is also known as a DNA marker.

GEL ELECTROPHORESIS USES Compare sizes of DNA to a known sample Identity of tissues Identity of criminal Identity of father in a paternity test Extract particular DNA fragment from gel