Sequencing DNA1. Maxam & Gilbert's method (chemical cleavage) 2. Fred Sanger's method (dideoxy method) 3. AUTOMATED sequencing (dideoxy, using fluorescent

tags)4. DNA-chip sequencing 5. Sequencing of individual bases (at the atomic level!)

APPLICATIONS OF DNA SEQUENCING: • Sequence information    • Identification of mutations. • Evolutionary relationships. •  Gene identity and mapping. • Gene expression questions

Maxam & Gilbert's method (chemical cleavage)

 

Maxam and Gilbert sequencing gel

Fred Sanger's method (dideoxy method)

 

Sanger method

Automated sequencing• almost all done this way today• basically Sanger method• 1400 bp from gel• ABI owns the labelled dideoxy technology• Licor sequences with labelled primers• Use different colour label for each

terminator base and can run all in same well on gel

• Laser scanner reads sequence, and sends the data to a computer

Automated sequencing• DNA replication reactions in a test tube, but in the

presence of trace amounts of all four of the dideoxy terminator nucleotides. A gel separates the resulting fragments by size and can 'read' the sequence from it, bottom to top.

• machine to run the gels and to monitor the different colors as they come off the bottom. It's called an automated DNA sequencer. There's an ultraviolet laser built into the machine that shoots through the gel near the botton and scans side to side, checking for bands of fluorescent colors to pass through its beam. There might be as many as 96 'lanes' of samples running in one gel.

• The four colors red, green, blue and yellow each represent one of the four nucleotides.

• This is a plot of the colours detected in a gel scanned from smallest fragments to largest. The computer even interprets the colors

• DNA microarray experiments are high throughput biological assays for measuring the abundance of DNA or RNA sequences in different types of cell samples for thousands of sequences simultaneously.

• Exploit the availability of sequence data to get information on gene expression in different types of cells.

• DNA microarrays rely on the hybridization properties of nucleic acids to monitor DNA or RNA abundance on a genomic scale in different types of cells.

• The ancestor of DNA and cDNA microarrays: the Southern and Northern blot.

Applications of microarrays

• Measuring transcript abundance (cDNA arrays);

• Genotyping;

• Estimating DNA copy number

• Determining identity by descent

• Measuring mRNA decay rates;

• Identifying protein binding sites;

• Determining sub-cellular localization of gene products

• Sequencing

DNA-chip sequencing -type of array-look for specific sequence presence-often used in gene expression experiments

http://upload.wikimedia.org/wikipedia/en/c/c8/Microarray-schema.jpg

http://www.dnalc.org/ddnalc/resources/chr11.html

time lapse of the DNA-Restriction Enzyme EcoP15I Complex obtained using fast-scan atomic force microscopy (AFM). Scale bar is 100 nm.Source: Crampton, N., et al. PNAS, 2007. (Click image for larger version)

Scanning EM of DNA

DF STEM of RNA