p.1
06. DNA sequencing
Primrose, Twyman & Old
Principles of Gene Manipulation, 6th edition (2002)
Chapter 7 (partim: pages 120-131)
Primrose & Twyman
Principles of Gene Manipulation and Genomics, 7th edition (2006)
Chapter 7 & pages (partim: pages 96-111 and 77-79 and 213-214)
- 1960-1975’s
- DNA polymerase reactions
- X174 : F. Sanger : plus-minus method
- Chain termination by dideoxynucleotides
(- chemical degradation method : Maxam & Gilbert)
not in 2007-2008
- Basics of the dideoxy-chain termination method
- copying single stranded template
- primer oligonucleotide
- generation of a nested set : unique 5' end
- separation according to chain length (PAGE) : n > < n+1
- labelling (detection of products) : 32P (-32P-dNTP)
- Problem aspects:
- fractionation : "gel compressions"
- fractionation : resolution (label, sensitivity)
- reading distance (bands in parallel lanes)
- unequal band intensities
- polymerase stops & pausing
G. Volckaert
DNA sequencing
12/02/2016
p.2
- Initial optimisation
- Klenow polymerase
=>
T7 DNA polymerase (Sequenase)
processivity, dNTP versus ddNTP
=>
Taq polymerase (higher temperature)
- control over gel temperature (avoiding "compressions"!)
- other labels (35S, 33P, non-isotopic) : sharper bands
-radiation
32
P
35
S
33
P
3
H
14
C
half-life
14.3 days
87.4 days
25 days
12.3 years
5,730 years
- primers : by chemical oligonucleotide synthesis
- template : cloning in M13 vectors (later on: in fasmids)
"universal" primer, master primer, forward & reverse primer
- Using synthetic primers: also double-stranded templates are allowed
compare: circular versus linear (presence of 3’ends !)
- PCR: cycle sequencing
25 cycli : denaturation step, annealing step and elongation/termination step
- Labelling :
- dNTP
(-phosphate position)
: many incorporations => high sensitivity
- primer
(5' end)
: single end-label => fewer side-products
- ddNTP
(-phosphate position)
: label by termination => false terminations invisible
- Compare: fixed moment vs on-line detection (real-time) vs trapping (Pohl)
G. Volckaert
DNA sequencing
12/02/2016
p.3
- "Automated" sequencing
Fluorescent labelling for detection (dyes)
- tagged primer : 1 tag, 4 reactions, 4 lanes
- more tags with different emission wavelengths : 4 tags, 4 reactions, 1 lane
- tagged ddNTPs : 4 tags, 1 reaction, 1 lane (is it substrate of the polymerase!!??)
- one lane :
- only "intra-lane" resolution is important
- fixed detectors versus scanning detector (idem for laser)
- corrections may be needed due to differences in dye properties
- on-line detection => PC => data processing & analysis
- ET-primers, Bodipy primers
- Examples (see figures)
- Novel equipment : fractionation by capillary electrophoresis : 1, 4, 8, 96 capillaries
50 m silica capillary; matrix is often linear polyacrylamide
Robotics
- Accuracy : - standard error frequency allowed 1/10000 – 1/100000 bp
- effect of error frequency on encoded gene products
- analysis on both strands is required
- preparation on single-stranded templates
- M13 vectors
(see chapter 4)
- fasmid (phasmid, phagemid) vectors
(see chapter 4)
- exonuclease III treatment
(see chapter 2)
- denaturation
- aPCR (asymmetric PCR) :
G. Volckaert
unequal primer concentrations (see chapter 5)
DNA sequencing
12/02/2016
p.4
- strand separation on “magnetic beads” after biotin labelling
- labelling at a single end
- filling-in asymmetric restriction ends
- PCR with one standard + one biotin-labelled primer
- immobilisation by streptavidin onto the magnetic beads
- strand separation by NaOH, then physical separation using a magnet
- adaptation of ends to use fluorescently-labelled primers
Other methods :
- Multiplex sequencing (G. Church & Kiefer-Higgins)
not in 2007-2008
- indirect detection by hybridisation onto the ladders
- Pyrosequencing (‘sequencing by synthesis’)
- pyrophosphate release during polymerase reactions
- pyrophosphate => ATP (APS + ATP-sulphurylase)
- ATP
[luciferase] => AMP + PPi + h
- relatively short distances (diagnostics, comparative sequencing/confirmation)
- SBH ('sequencing by hybridisation') (R. Drmanac)
- sequence complexity :
8 => 65536
256 x 256
- DNA immobilised on matrix, labelled oligonucleotides as probes
=> multiple hybridisation rounds
or:
- reverse blots: immobilised oligonucleotides, labelled DNA fragment as probe
=> limited hybridisation rounds
- array format :
at 0,1 mm : 2,56 x 2,56 cm
at 0,04 mm : 1 x 1 cm
G. Volckaert
DNA sequencing
12/02/2016
p.5
- DNA chips : preparation by in situ synthesis (see chemical DNA synthesis)
or direct application of the oligonucleotides
- microtiter format
12 x 8
=
96
x4
384
x4
1456
- macro-arrays, micro-arrays : => robotics
- Feasibility: possible with segments of 200 or a few hundreds of bp. Particularly
efficient for comparative (control) (re-)sequencing or analyses of small
changes (mutants, polymorphisms, SNPs).
- Maldi – TOF
- analysis of nested set by mass spectrometry
Analysis of larger DNA’s (> 1 kb)
- definition :
- CONTIG
- “reverse sequencing”
- 3 strategies
- SHOTGUN : accumulation of data into contigs
- redundancy : => remaining “gap”s
- both strands
- routine phase and “finishing” phase
- systematic deletions : “progressive sequencing”
- method of Henikoff (+ variations thereoff) : using exonuclease III
- importance of polylinker
- 5’-protruding, 3’-protruding, blunt : protecting one end from degradation
- exonuclease III + nuclease S1 or mung-bean nuclease or exonuclease VII
G. Volckaert
DNA sequencing
12/02/2016
p.6
- “primer walking” sequencing
- depends on large capacity of oligonucleotide synthesis
- time lags between consecutive cycles
- at genome scale : currently several tens of entire genomes have already been sequenced
- “whole genome shotgun” approach :
=> computing power ! repeated sequences !
- “minimal tiling path” approach : “hierarchical approach” :
- metagenome sequencing
Novel and/or upcoming approaches
(as illustration only, not exhaustive)
- 454 sequencing
- supported oligonucleotide detection (SOLiD)
- single-molecule sequencing
- nanopore sequencing
- optical trapping
- polony sequencing
G. Volckaert
DNA sequencing
12/02/2016