Dideoxy Chain Termination Method

agarose gel electrophoresis

analyzes DNA fragments in size range 100-20000 bp

pulsed field gel electrophoresis (PFGE)

analyzes DNA fragments greater than 20,000 bp

PFGE process

electrophoretic equipment used to vary direction and intensity of the electric field at fixed interval; with each change, long DNA molecules reorientate to facilitate movement through gel pores; separation depends on rate of reorientation and rate of migration; runs for 18-24 hours

polyacrylamide gel electrophoresis (PAGE)

used to separate smaller nucleotides (10-4000 bp)

polyacrylamide gel

gel formed by polymerization and cross-linking monomer; gel has smaller pore than agarose allowing for more effective molecular sieving; usually 50-60 cm long, 20-50 cm wide, and 0.4 mm thick

preparation of polyacrylamide gel

acrylamide monomer, bis-acrylamide cross-linker, urea, and tris-borate buffer are mixed in a beaker; the polymerization catalysts are added to the mixture and the mixture is poured between two glass plates separated by spacers and sealed at the base

PAGE process

power pack applies a potential difference of ~1500 V across the gel, the DNA migrates towards the positive electrode; aluminum plates positioned against gel to spread out heat and prevent glass cracking; heat and urea ensure that the DNA remained denatured and single stranded

ethidium bromide

a stain that can be used to stain the polyacrylamide gel to detect DNA bads

autoradiography of PAGE

DNA is radioactively labeled before it is separated; the gel is dried on a thin white cardboard and exposed to x-ray film in the dark; the B-particles from the radioactive DNA blackens the film; the film is developed and allows you to detect DNA bands

Sanger Sequencing

sequencing of DNA using the dideoxy chain termination method

dideoxynucleotides (ddNTPs)

nucleotides that base pair normally but do not have a 3’ hydroxyl group, so they terminate chain extension

components of sanger sequencing solution

template DNA, oligonucleotide primer to anneal to template, DNA polymerase, all 4 nucleotides (one being the ddNTP); need four solutions, each with a different ddNTP

Sanger Sequencing method

1. DNA template is denatured by heating and the primer is allowed to anneal

2. Solution is split into four vials and DNA polymerase and all four nucleotides are added to each

3. Add a different ddNTP to each vial

4. the polymerases lengthen the primers; stop when a ddNTP is added

5. The resulting chains have lengths that end at each nucleotide of the ddNTP in the vial

6. reaction mixtures are separated by high resolution page and the DNA bands are identified by autoradiography

7. the autoradiographs are used to piece together the DNA sequence

High Resolution PAGE

PAGE that can separate chain lengths that differ by only one nucleotide

fluorescent sanger sequencing

different fluorescent dyes are attached to each ddNTP; all ddNTPs are added to one vial; products are run in PAGE and a detector detects the sequence by determining the fluorescence that runs of; they will run off in order of their chain length!

large scale DNA sequencing

one run of sanger sequencing can only sequence about 800 bases in each run; when running bigger DNA sequences, the DNA is split into 1 kb fragments; when enough fragments are sequenced, a computer program is used to compile overlapping sequences into one long sequence

Bioinformatic analysis of long DNA sequences

computer programs used to ID open reading frames (start with a start codon and end with a stop codon); sequence alignments carried out to identify open reading frames that are homologous to genes of known functions; other programs can be used to predict protein structures from DNA sequences

advantages of dideoxy sequencing method

a long read of about 900 bases can be obtained from each template; accurate and well-tried; still best approach for sequencing regions of DNA less than 50 kb

disadvantages of dideoxy sequencing method

scale up for ‘massively parallel’ sequencing is uneconomic