DNA SEQUENCING DNA SEQUENCING

Principles & methods Principles & methods

Topic Outline

Running the reaction of all the dideoxy

nucleotides using different dyes

generates this type of diagram in same lane.

Sometimes the spacing Sometimes the spacing between the bands is between the bands is hard to measure.hard to measure.

Thus use machine to run Thus use machine to run and read the and read the electrophoresis.electrophoresis.

Capillary electrophoresis:Capillary electrophoresis: the fragments are piped the fragments are piped through a tiny glass-fiber through a tiny glass-fiber capillary during the capillary during the electrophoresis step, and electrophoresis step, and they come out the far they come out the far end in size-order.end in size-order.

Chemical cleave methodChemical cleave method Sequence small Sequence small

fragments of DNAfragments of DNA The radioactive labelling The radioactive labelling

is done on the dsDNA.is done on the dsDNA. Division of aliquots is Division of aliquots is

done by methylation or done by methylation or removal of base.removal of base.

Requires DNARequires DNA Breaks DNA at different Breaks DNA at different

nucleotidesnucleotides

Enzymatic cleave methodEnzymatic cleave method Sequencing small Sequencing small

fragments are fragments are problematic.problematic.

The radioactive labelling The radioactive labelling is done on the ssDNA.is done on the ssDNA.

Allow high throughput Allow high throughput automated sequencing automated sequencing techniques. techniques.

Allow Real Time detection. Allow Real Time detection. Requires DNA synthesisRequires DNA synthesis Termination of chain Termination of chain

elongationelongation

Comparison of Southern, Northern, and Western analyses of Gene X

SOUTHERN BLOTTING

The technique was developed by E.M. Southern in 1975.

The Southern blot is used to detect the presence of a particular piece of DNA in a sample.

The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

Southern hybridization

Transfer buffer

Detection of an RFLP by Southern blotting

Flow chart of Southern hybridizationPreparing the samples and running the gel

Southern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

IsotopeNon-isotope

SOUTHERN BLOTTING

The key to this method is hybridization.

Hybridization-process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

SOUTHERN BLOTTING

There are 2 important features of hybridization: The reactions are specific-the probes will

only bind to targets with a complementary sequence.

The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

Comparison of Southern, Northern, and Western blotting techniques

Southern blotting Northern blotting Western blotting Molecule detected

DNA (ds) mRNA (ss) Protein

Gel electrophoresis

Agarose gel Formaldehyde agarose gel

Polyacrylamide gel

Gel pretreatment

Depurination, denaturation, and

neutralization

- -

Blotting method Capillary transfer Capillary transfer Electric transfer Probes DNA

Radioactive or nonradioactive

cDNA, cRNA Radioactive or nonradioactive

primary antibody

Detection system

Autoradiography Chemiluminescent

Colorimetric

Autoradiography Chemiluminescent

Colorimetric

Chemiluminescent Colorimetric

MB206

What Is a Mutation?

Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine (C), a mutation is a change in the order of these nucleotides.

A change in the order can cause the gene to encode for wrong proteins and inhibit the function of the gene or cause the gene to be virtually inactive.

Mutagenesis

Mutagenesis (the creation or formation of a mutation) can be used as a powerful genetic tool. By inducing mutations in specific ways and then observing the phenotype of the organism the function of genes and even individual nucleotides can be determined.

Some types of Mutagenesis Directed Mutagenesis

Site-directed/Site-specific Mutagenesis

Mismatched Mutagenesis