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DNA sequencing
Determination of nucleotide sequence the determination of the precise sequence of nucleotides in a sample of DNA
Two similar methods: 1. Maxam and Gilbert method 2. Sanger method They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined
MC chapter 12
Maxam-Gilbert
Walter Gilbert
Harvard physicist Knew James Watson Became intrigued with the biological side Became a biophysicist
Allan Maxam
Maxam-Gilbert Technique
Principle Chemical Degradation of Pyrimidines
Pyrimidines (C, T) are damaged by hydrazine Piperidine cleaves the backbone 2 M NaCl inhibits the reaction with T
The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified
G C
G C
A T
32p
polynucleotide kinase
Chemical cleavage Method (Maxam & Gilbert 1980) End labeled DNA
Depurination &
CH3
N C
C CH N
Bond breakage
G
N
Sugar
A G T C
A G T C
4 6 4/3
G (4)
T C G A G G A T G
A + G (6)
C + T (3)
C (1)
G
T
A+G
C+T
C
G A G G A T G
Requires lots of purified DNA, and many intermediate purification steps Relatively short readings Automation not available (sequencers) Remaining use for footprinting (partial protection against DNA modification when proteins bind to specific regions, and that produce holes in the sequence ladder)
In contrast, the Sanger sequencing methodology requires little if any DNA purification, no restriction digests, and no labeling of the DNA sequencing template
Sanger Method
Fred Sanger, 1958
Was originally a protein chemist Made his first mark in sequencing proteins Made his second mark in sequencing RNA
Sanger Method
in-vitro DNA synthesis using terminators, use of dideoxinucleotides that do not permit chain elongation after their integration DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system Incorporation of di-deoxynucleotides into growing strand terminates synthesis Synthesized strand sizes are determined for each dideoxynucleotide by using gel or capillary electrophoresis Enzymatic methods
Dideoxynucleotide
PPP O
5 CH2 O BASE
3
no hydroxyl group at 3 end prevents strand extension
The principles
Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP Separate by gel electrophoresis Read DNA sequence
3 primer 5
CCGTAC 5 3 dNTP
ddCTP ddGTP
ddATP ddTTP
GGCA
A
GGCAT
T C G
GGC
G GG GGCATG
Separation Detection
ddC
dN : ddN 100 : 1
Dideoxy sequencing relies on copying a single fragment of DNA many times but each copy is prematurely terminated.
This produces a series of nested DNA copies, each differing by a single base in length.
These fragments are separated on a polyacrylamide gel and the DNA sequence is determined by reading the terminating base of each fragment on the gel
A G T C
ddNTP
Dideoxynucleoside triphosphates lack an -OH group at the 3-carbon position and cannot add another nucleoside at that position, thus preventing further DNA synthesis
-OH
-OH
-OH
-OH
C
fluorescent dyes
G G
-OH
T T
-OH
C C
-OH
T T
-OH
G A G C C T G G A G C
-OH -OH -OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
Chain-Terminator Method (Sanger, 1977) DNA fragment released The DNA copy is released and the original DNA strand is available to be copied again
Electrophoresis
Spacing plates
Template DNA
A G T C
Electrophoresis
Fluorescent bands after electrophoresis
The fluorescent bands in each lane of the DNA are read from the bottom up to determine the sequence of the DNA segment.
Gel bottom up sequence Complementary sequence
G A T C T T G C G T C A G T C A
A G T C
Electrophoresis
Fluorescent bands after electrophoresis
The fluorescent bands in each lane of the DNA are read from the bottom up to determine the sequence of the DNA segment.
Electrophoresis
Fluorescent bands after electrophoresis
The fluorescent bands in each lane of the DNA are read from the bottom up to determine the sequence of the DNA segment.
Electrophoresis
Documentation
A printout of the laser scan is also prepared as a permanent record
Electrophoresis
Template
ssDNA vectors
M13 pUC
Primers
Universal primers
cheap, reliable, easy, fast, parallel BULK sequencing
Custom primers
expensive, slow, one-at-a-time ADAPTABLE
Extension
Polymerase
Sequenase Thermostable (Cycle Sequencing)
Terminators
Dye labels (Big Dye)
spectrally different, high fluorescence
Separation
Gel Electrophoresis Capillary Electrophoresis
suited to automation
rapid (2 hrs vs 12 hrs) re-usable simple temperature control 96 well format
Sample Output
1 lane
Comparison
Sanger Method
Enzymatic Requires DNA synthesis Termination of chain elongation
Sequencing done by TIGR (Maryland) and The Sanger Institute (Cambridge, UK) Here we report an analysis of the genome sequence of P. falciparum clone 3D7, including descriptions of chromosome structure, gene content, functional classification of proteins, metabolism and transport, and other features of parasite biology.
Sequencing strategy A whole chromosome shotgun sequencing strategy was used to determine the genome sequence of P. falciparum clone 3D7. This approach was taken because a whole genome shotgun strategy was not feasible or cost-effective with the technology that was available at the beginning of the project. Also, high-quality large insert libraries of (A T)-rich P. falciparum DNA have never been constructed in Escherichia coli, which ruled out a clone-by-clone sequencing strategy. The chromosomes were separated on pulsed field gels, and chromosomal DNA was extracted
The shotgun sequences were assembled into contiguous DNA sequences (contigs), in some cases with low coverage shotgun sequences of yeast artificial chromosome (YAC) clones to assist in the ordering of contigs for closure. Sequence tagged sites (STSs)10, microsatellite markers11,12 and HAPPY mapping7 were also used to place and orient contigs during the gap closure process. The high (A /T) content of the genome made gap closure extremely difficult79. Chromosomes 15, 9 and 12 were closed, whereas chromosomes 68, 10, 11, 13 and 14 contained 337 gaps (most less than 2.5 kb) per chromosome at the beginning of genome annotation. Efforts to close the remaining gaps are continuing.
Methods: Sequencing, gap closure and annotation The techniques used at each of the three participating centres for sequencing, closure and annotation are described in the accompanying Letters79. To ensure that each centres annotation procedures produced roughly equivalent results, the Wellcome Trust Sanger Institute (Sanger) and the Institute for Genomic Research (TIGR) annotated the same100-kb segment of chromosome 14. The number of genes predicted in this sequence by the two centres was 22 and 23; the discrepancy being due to the merging of two single genes by one centre. Of the 74 exons predicted by the two centres, 50 (68%) were identical, 9 (2%) overlapped, 6 (8%) overlapped and shared one boundary, and the remainder were predicted by one centre but not the other. Thus 88% of the exons predicted by the two centres in the 100-kb fragment were identical or overlapped.
Previous sequencing techniques: one DNA molecule at a time Needed: many DNA molecules at a time -- arrays
Sequence by DNA polymerase -dependent chain extension, one base at a time in the presence of a reporter (luciferase) Luciferase is an enzyme that will emit a photon of light in response to the pyrophosphate (PPi) released upon nucleotide addition by DNA polymerase Flashes of light and their intensity are recorded
B
The readout is recorded by a detector that measures position of light flashes and intensity of light flashes
From www.454.com
MW spectrum of 33-mer 5-ACT AAT GGC AGT TCA TTG CAT GAA TTT TAA AAG-3
One early rationale for developing hybridization arrays was de novo sequencing. As originally conceived, this strategy [sequencing by hybridization (SBH)] involved annealing a labeled unknown DNA fragment to a complete array of short oligonucleotides (e.g. all 65,336 combinations of 8-mers) and deciphering the unknown sequence from the annealing pattern. Over the past decade, SBH has largely been eclipsed by the use of DNA arrays for single nucleotide polymorphisms (SNP) and expression analysis. This is partly due to the amount of diagnostic or biological information to be gained per feature on the array. For example, expression monitoring of the entire human genome could be performed using a microarray composed of 100,000 gene-specific sequences (or possibly many fewer), whereas the same number of features would allow resequencing of only 25,000 bases. Another stumbling block for sequencing applications is the use of short oligonucleotide probes. These present such problems as ambiguous reads as-sociated with repeat regions within the unknown target sequence, formation of secondary structures in some oligonucleotide probes that result in little or no detectable signal, and hybridization of oligonucleotides with single mismatches (false positives), which can be especially common at the terminal base pair.
DNA Sequencing by Hybridization This strategy involved annealing a labeled unknown DNA fragment to a complete array of short oligonucleotides (e.g. all 65,336 combinations of 8-mers) and deciphering the unknown sequence from the annealing pattern.
Principle of Pyrosequencing
(http://www.pyrosequencing.com/pages/technology.html)
Pyrosequencing is to sequence DNA by enzymatic DNA synthesis, and the DNA sequence is determined the from the signal peak of released photons during the synthesis. It includes the following 5 steps: Step 1 A sequencing primer is hybridized to a single stranded, PCR amplified, DNA template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5 phosphosulfate (APS) and luciferin. Step 2 The first of four deoxynucleotide triphosphates (dNTP) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.
Step 3 ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5 phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram. Each light signal is proportional to the number of nucleotides incorporated.
Step 4 Apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added.
Step 5 Addition of dNTPs is performed one at a time. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPaS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DN polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the pyrogram.
Summary of Pyrosequencing Pyrosequencing is to sequence DNA by enzymatic DNA synthesis, and the DNA sequence is determined the from the signal peak of released photons during the synthesis.
Zipper-sequencing of DNA A DNA construct was engineered such that one of its extremities had one strand anchored to a surface via a long DNA fragment, and the other strand was bound to a small bead, itself stuck to a flexible glass fiber used as a force sensor. As the surface is displaced, the molecule is unzipped and the force to unpair two bases measured by the force sensor.
DNA
E
2nm
Off-lattice simulations
A Sequencing Array
Conclusions
1. The traditional Sanger method of sequencing DNA is slow, costly, and inaccurate. It takes about 15 years to sequence human DNA and costs about 3 billion US dollars. The overlap of predicted novel gene sets between Celera and Ensembled is about 20%. 2. The new method by nanopore sequencing can be fast, inexpensive, and accurate. It takes about 1 day to sequence 100 million bases by using a sequencing array and high accuracy can be achieved by analyzing several time series. 3. The translocation time of polynucleotide chains is well controlled by the frequency of the rotating electric field. Specifically, it increases linearly with the rotating period for frequency less than 10 KHz. 4. The translocation time of each nucleotide is quantized in unit of a quarter of rotating period, which can be used to predict the sequence accurately.