DNA Sequencing Lecture Notes

DNA Sequencing Lecture Notes http://www.dnalc.org/ddnalc/resources/sangerseq.html http://www.dnalc.org/ddnalc/resources/cycseq.html On board: Explain Sanger sequencing method • Based on ordinary DNA synthesis using a DNA polymerase (usually Klenow fragment of E. coli DNA pol) • Components of the reaction: 1. Single stranded template or target DNA 2. Sequence-specific primer (specificity affected by temp, concentration, etc. like PCR) 3. DNA polymerase 4. All 4 dNTPs (IMPORTANT) 5. ONE of the 4 dideoxynucleotide triphosphates (ddATP, or ddCTP, or ddTTP, or ddGTP) • Incorporation of ddNTP is random; when it occurs, chain termination • Because there are many copies of the template being read simultaneously, a big random mix of newly synthesized DNA products is made, of all sizes, each ending in the ddNTP of that reaction • Traditionally, this mix of DNAs is run on a (denaturing) polyacrylamide gel which can resolve bands differing in size by as little as 1 nt • If synthesized DNA was radioactively labeled, then each ddNTP reaction is run in a separate lane on the gel, and the sequence is read 5’ to 3’ from the bottom of the gel up. Polyacrylamide gels: • Poured as liquid with a catalyst added which crosslinks the acrylamide into polyacrylamide, irreversibly solidifying it (can’t melt & re-pour like agarose) • Very thin gels (1-2 mm), usually poured between two glass plates • Much higher resolution of fragment sizes than agarose; can easily separate DNA fragments that differ in size by only one nucleotide • (Fragment sizes separated in this way are small, from about 20-300 nucleotides) • Polyacrylamide sequencing gels usually contain urea as a denaturing agent, so DNA runs single-stranded Automated sequencing: • Each ddNTP is fluorescently labeled with a different “color” • All reactions can be run together, again generating a mix of all DNA fragment sizes, each ending in a ddNTP • Products are separated by size (1 nt difference) and analyzed by a laser & detector. • Data are presented as a chromatogram showing fluorescence intensity and color of each DNA fragment. • Chromatogram sequence is read 5’ to 3’, from left to right. Point out that relatively higher concentration of ddNTP in reaction will give sequence read relatively closer to the primer (as ddNTP incorporation is more likely earlier). Conversely, a relatively lower [ddNTP] gives longer reads (further away from primer). Show animations: http://dnalc02.cshl.edu/ddnalc/resources/sangerseq.html (sangerseq movie, downloaded) http://dnalc02.cshl.edu/ddnalc/resources/cycseq.html (automated seq movie, downloaded) Discuss shotgun sequencing • A strategy for sequencing large DNAs (uses Sanger method) • A single Sanger method reaction can give you about 200-500 nt of sequence • To continue sequencing a longer DNA, you would need to then synthesize a sequence-specific primer at the end of the sequence you just read to start the next Sanger reaction • If your DNA is a Mb-size chromosome, this would take forever. • Shear your big DNA randomly into small (~2 kb) fragments • Clone MANY of these fragments into plasmids (one fragment per plasmid) and transform them into bacteria to make a library • Use two “universal” sequencing primers, which hybridize to the plasmid on either side of the insertion site, to sequence into the insert (your cloned small piece of the big DNA) • If you sequence enough clones, you’ll get sequence data for the whole original DNA • It is important to sequence both strands (i.e., from both ends of the insert) to maximize accuracy as certain regions of ssDNA sequence poorly • Because the DNA was originally broken up at random, many of the clones should overlap. • Use computer to find regions of overlap and reassemble all the small sequence bits into one big long sequence for the original molecule. This reassembled puzzle is called a contig. • If there are gaps in your contig (regions with no overlap), you can either choose more clones to sequence at random, or, design sequence-specific primers to sequence into the gap Show animation (on the web): http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/humanShot.swf