Sequencing

– 2 main types:

dideoxy (Sanger) sequencing

– interrupted DNA synthesis by DNA polymerases

chemical (Maxam-Gilbert) – partial DNA hydrolysis

dideoxy (Sanger) sequencing

– most commonly used technique

– based on extension of oligonucleotide primers by DNA polymerase

– extension stopped by dideoxy nucleotides (ddNTP)

– lack 3' OH (Fig. 3.4)

– cannot form phosphodiester bond to 5' PO₄

– always form end of new strand

traditional procedure: do 4 reactions

– each contains template DNA, primer, 4 dNTPs, 1 ddNTP

– ddNTP differs in each reaction – (limited amount)

(ddATP, ddCTP, ddGTP, ddTTP)

– each reaction stops at different nucleotides

– get mix of different-sized molecules (Fig. 3.5)

– all ended at same type of base (for each reaction)

– run 4 reactions on polyacrylamide gel (Fig. 3.6)

– DNA molecule – 1 nucleotide length difference separated

– smaller molecules run farther

– so each step up ladder is 1 nucleotide longer

– can read sequence off gel

components of reaction:

template DNA

– best template is single-stranded DNA

– no denaturation step before primer annealed

– clones in M13 vectors

– used by labs that do lots of sequencing

– process can be automated

– prepare M13 clones in 96-well microtitre plates

– do sequencing reactions in plates

– load on 96-well gels or capillary apparatus

– transfers can be done by robot equipment

– up to 5000 sequencing reactions per week

– double stranded template can also be used

– plasmid DNA or PCR product

– denatured by alkali or heat

– more problems with primer annealing & extension

– but no special vectors needed

primers – 15-25 base oligonucleotides

– similar to PCR primers – but only 1 per reaction

– often use universal primers

– vector-specific primers

– match sequences near multiple cloning site

– work for all clones in same vector

– sequence from ends of cloned DNA

– e.g. T3, T7 promoter region of pBluescript

DNA polymerase – need enzyme without 5'-3' exonuclease

– so primer extension products not degraded

– 1^st enzyme used – Klenow fragment of E. coli DNA pol I

– poor processivity in vitro – falls off template early

– improved results with Sequenase

– modified T7 bacteriophage DNA polymerase

– 3'-5' exonuclease deleted from cloned gene

– high processivity – incorporates ddNTPs efficiently

– modified Taq polymerases

– 5'-3' exonuclease deleted from cloned gene

– mutations to allow efficient ddNTP usage

– used for cycle sequencing – like PCR

– multiple cycles of denaturation, annealing, extension

– get lots of product from less template

modified dNTPs (optional) – help resolve compressions

(problems caused by secondary structure in DNA)

– found in high GC regions

– stops polymerase or changes mobility of DNA

– get bands running close together or BAFLs

(Bands in All Four Lanes)

– modified bases (dITP, 7-deaza-dGTP)

– 2 H-bonds with dCTP – weaker secondary structure

– other solutions

– cycle sequencing (heat melts secondary structure)

– sequence in both directions

– secondary structure different in other direction

– also allows confirmation of 1^st strand sequence

– formamide and urea in gels

– denatures secondary structure

labelled nucleotides – radiolabel or fluorescent dye

– allows primer extension products to be seen in gel

3 ways of labelling:

1. end-labelling of primer

– using gamma-labelled ATP & polynucleotide kinase

– or fluorescent label on primer

advantage: – easy to do

disadvantages:

– single label per molecule – fainter signal

– all molecules labelled

– whether dideoxy terminated or not

2. incorporation of radiolabel

– alpha-labelled dNTP in reaction mix

advantages: – easy to do

– multiple labels per molecule – stronger signal

disadvantages: – all molecules labelled

– whether dideoxy terminated or not

– does not work with fluorescent labels

3. labelled ddNTPs

– e.g. Amersham kit

– ³³P-labelled ddNTPs

– label only incorporated in dideoxy-terminated DNA

– but get weaker signal – 1 label per molecule

– or dye terminator kits

– fluorescent dye attached to ddNTPs

advantages:

– can attach different fluorescent dyes to each ddNTP

– run all 4 reactions in same lane of gel

– computer sorts out base by colour of fluorescence

disadvantages:

– not all polymerases will incorporate modified ddNTPs

4 types of label:

1. ³²P – stronger beta-particle emitter – need thick shielding

– gives visible results with very little label

– e.g. on Southern blots

– but too strong for sequencing

– bands near top of gel close together

– strong ³²P signal produces overlap

– sequence unreadable after 100 bases

– also short half life – DNA degraded as ³²P decays

2. ³³P – weaker beta-particle emitter

– gives readable sequence for >200 bases

– also has longer half life

3. ³⁵S – incorporated in alpha PO₄ – replaces 1 O

– weaker beta-particle emitter than ³³P

– gives readable sequence for >200 bases

– but longer exposure times needed

– has longer half-life than ³³P

4. fluorescent labels

– glow different colours when excited with laser

– 4 reactions in 1 tube/gel lane– if use dideoxy label

– used for automated sequencing

– results read by computer – as bands run through gel

– each sequencing reaction run through entire length of gel

– get good separation of higher molecular weight bands

– > 400 bases of good sequence per reaction

(> 600 bases with optimal equipment & conditions)

– preferred technique in most labs

chemical (Maxam-Gilbert) sequencing

– end-label DNA molecule at one end only

– similar to procedure for restriction map

– treat with chemicals that cause cleavage at specific bases

– 5 reactions – cleave at G, A+G, C+T, C, A>C

– use conditions that produce ~1 cleavage per molecule

– each reaction has mix of different sized products

– only end-labelled products visible on gel

– get ladders of bands in gel – differ by 1 base

– read results by comparing lanes on gel

– rarely used for general sequencing

– labour intensive, toxic chemicals

– shorter sequences (³²P end-label)

– used for specialized purposes:

– sequencing oligonucleotides

– DNA footprinting

– finding binding sites for DNA-binding proteins – bind protein to DNA & chemical sequence

– compare control to protein-protected DNA

– see gap in bands

– protein protected DNA from cleavage

– also used to find methylation patterns

– methylated C is not cleaved like normal C