Recombinant Tech II

DNA MAPS

Genetic = recombination frequencies. = recombination frequencies.Cytological = banding pattern of chromosomes= banding pattern of chromosomesPhysical= Distances in bp. kb, megabase pairs.= Distances in bp. kb, megabase pairs.

BLOTS

SouthernSouthern NorthernNorthern WesternWestern

SOUTHERN

M.E Southern ? Presence of restricfion fragments of DNA in ? Presence of restricfion fragments of DNA in

a samplea sample Involves separation, transfer and hybrdizationInvolves separation, transfer and hybrdization Single gene/ larger piece of DNA.Single gene/ larger piece of DNA.

Process

1) DNA is 1) DNA is digested with a RE, with a RE, separated by gel by gel electrophoresis. electrophoresis.

2) 2) Transferred to a membrane. Same pattern. to a membrane. Same pattern. 3) The blot is 3) The blot is incubated with many copies of a probe. with many copies of a probe.

This probe will form base pairs with its This probe will form base pairs with its complementary DNA sequence and bind to form a ds complementary DNA sequence and bind to form a ds DNA molecule. Radioactive/ enzyme bound (ALP or DNA molecule. Radioactive/ enzyme bound (ALP or horseradish per). horseradish per).

4) The location of the probe is revealed by incubating 4) The location of the probe is revealed by incubating it with a substrate = colored product.it with a substrate = colored product.

Blotting

Nitrocellulose/Nylon paper laid over the gelNitrocellulose/Nylon paper laid over the gel The gel is supported on a layer of sponge in a The gel is supported on a layer of sponge in a

bath of alkali solutionbath of alkali solution Buffer is sucked through the gel and the Buffer is sucked through the gel and the

nitrocellulose papernitrocellulose paper As the buffer is sucked through, it denatures As the buffer is sucked through, it denatures

the DNA and transfers the single-stranded the DNA and transfers the single-stranded fragments from the gel to the surface of the fragments from the gel to the surface of the nitrocellulose sheet. Adhere.nitrocellulose sheet. Adhere.

Hybridization The nitrocellulose sheet is peeled off the gel.The nitrocellulose sheet is peeled off the gel. The sheet containing the bound ss DNA fragments is The sheet containing the bound ss DNA fragments is

placed in a sealed plastic bag together with buffer placed in a sealed plastic bag together with buffer containing a probe specific for the required DNA containing a probe specific for the required DNA sequence. Exposure.sequence. Exposure.

The sheet is removed from the bag and washed. Only The sheet is removed from the bag and washed. Only probe molecules that have hybridized to the DNA on probe molecules that have hybridized to the DNA on the paper remain attached. the paper remain attached.

Autoradiography = the DNA that has hybridized to Autoradiography = the DNA that has hybridized to the labeled probe will show up as bands on the the labeled probe will show up as bands on the autoradiograph.autoradiograph.

Closer look

Gel electrophoresis

Transfer of the DNA to a Membrane

The DNA is denatured –The DNA is denatured – Transferred onto a Transferred onto a

membrane. membrane. BlottedBlotted

Hybridization

Incubated with a solnIncubated with a solncontaining a probe. Comp.containing a probe. Comp.

A wash step removes anyA wash step removes anyprobe which is not tightlyprobe which is not tightlybound to the DNA on thebound to the DNA on themembrane. membrane.

Only DNA that matchesOnly DNA that matchesexactly will remain boundexactly will remain bound

Northern blotting

RNA molecules of varying lengths are RNA molecules of varying lengths are separated by size and blotted onto separated by size and blotted onto nitrocellulose. nitrocellulose.

A DNA probe (often a cDNA) is then used to A DNA probe (often a cDNA) is then used to identify bands that contain particular identify bands that contain particular sequences.sequences.

Useful for determining the conditions under Useful for determining the conditions under which specific genes are being expressed, which specific genes are being expressed, including which tissues express which genes ?including which tissues express which genes ?

Western Blot

Proteins are separated by electrophoresis and Proteins are separated by electrophoresis and blotted onto an appropriate support matrix. blotted onto an appropriate support matrix.

The matrix is then exposed to an antibody to The matrix is then exposed to an antibody to the desired protein and all unbound antibody is the desired protein and all unbound antibody is washed off. washed off.

The bands are detected by the binding of a The bands are detected by the binding of a second antibody that is radioactively labeled second antibody that is radioactively labeled and specific for the first antibody.and specific for the first antibody.