Lecture 19- sequencing

why sequence DNA

-all genes available for an organism to use: important for biologists

-not just sequencing, but positioning of genes and sequences of regulatory regions

-new recombinant DNA constructs must be sequenced to verify construction or positions of mutations

methods of sequencing

-sanger dideoxy

-maxam-gilbert chemical cleavage

-pyrosequencing

Sanger dideoxy (primer extension/chain termination) method

-most popular protocol for sequencing, very adaptable, scalable to large sequencing projects

maxam-gilbert chemical cleavage method

-DNA is labelled and then chemically cleaved in a sequence-dependent manner

-not easily scaled and tedious

pyrosequencing

-measuring chain extension by pyrophosphate monitoring

for dideoxy sequencing you need

1.single-stranded DNA template

2.A primer for DNA synthesis

3.DNA polymerase

4.Deoxynucleoside triphosphates and dideoxynucleotide triphosphates (ddA, ddT, ddG, ddC)

-each base generates different length of DNA, need to separate them

-chain terminates without 3’ hydroxyl, cannot add another nucleotide without it

primers for DNA sequencing

-oligonucleotide primers can be synthesized by phosphoramidite chemistry- usually designed manually then purchased

-sequence of the oligo must be complimentary to DNA flanking sequenced region

-oligos are usually 15-30 nucleotides in legnth

DNA templates for sequencing

-single stranded DNA isolated from recombinant M13 bacteriophage containing DNA of interest

-double-stranded DNA that has been denatured

-non-denatured double stranded DNA (cycle sequencing)

obtain single straded DNA from a double stranded source

-prime bottom strand with magnetic beads

-separate the magnetically-labelled strands

Reagents for sequencing DNA polymerase

-should be highly processive and incorporate ddNTPs efficiently

-should lack exonuclease activity

-thermostability required for “cycle sequencing”

sanger-dideoxy sequencing: basic method

a.) anneal the primer

b.) extend the primer with DNA polymerase in the presence of all four dNTPs, with a limited amount of a dideoxy NTP (ddNTP)

-DNA pol incorporates ddNTP in a template-dependent manner, but it works best if the DNA pol lacks 3’ to 5’ exonuclease activity

-ddATP in the reaction: anywhere there’s a T in the template strand, occasionally a ddA will be added to the growing strand

how to visualize DNA framents

-radioactivity: radiolabeled primers (kinase with 32P), radiolabelled dNTPS (gamma 35S or 32P)

-flourescence: ddNTPs chemically synthesized to contain fluors, each ddNTP fluorescens at a different wavelength allowing identification

Analysis of sequencing products

-polyacrylamide gel electrophoresis: good resolution of fragments differing by a single dNTP

-slab gels: as previously described

-capillary gels: require only a tiny amount of sample to be loaded, run much faster than slab gels, best for high throughput sequencing

cycle sequencing: denaturation occurs during temp cycles

-94 C DNA denatures

-45 C primer anneals

-60-72 C thermostable DNA pol extends primer

-repeat 25-35 times

-advantages: don’t need a lot of template DNA

-disadvantages: DNA pol may incorporate ddNTPs poorly

sequencing a genome

-break up the genome and put it back together

-assemble sequences by matching overlaps

sequencing large pieces of DNA: the shotgun method

-break DNA into small pieces (1000bp)

-clone pieces of DNA into M13

-sequence enough M13 clones to ensure complete coverage

-assemble genome through overlap analysis using computer algorithms, also polish sequences using mapping info from individual clones, characterized genes and genetic markers

-assisted by robotics

pyrosequencing

-cut a genome to DNA fragments 300-500bp long

-immobilize single strands on a very small plastic bead (1 piece of DNA per bead)

-amplify the DNA on each bead to cover each bead to boost the signal

-separate each bead on a plate with up to 1.6 million wells

sequencing by DNA polymerase

-dependent chain extension, one base at a time in the presence of a reporter (luciferase)

luciferase

-an enzyme that will emit a photon of light in response to the pyrophosphate (PPi) released upon nucleotide addition by DNA pol

-flashes of light and their intensity are recorded

Extension with individual dNTPs gives a readout

-the readout is recorded by a detector that measures position of light flashes and intensity of light flashes

-height of peak indicates the number of dNTPs added