DNA Sequencing
Sequencing means finding the order of nucleotides on a piece of DNA . Nucleotide order determines amino acid order, and by extension, protein structure and function (proteomics) . An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a genetic disorder . DNA sequence is important to detect the type of mutations in genetic diseases and offer hope for the eventual development of treatment DNA .
Methods of sequencing there are two main methods of DNA sequencing: 1-Sanger dideoxy (primer extension/chain-termination) method: most popular protocol for sequencing, very adaptable, scalable to large sequencing projects 2-Maxam-Gilbert chemical cleavage method: DNA is labelled and then chemically cleaved in a sequencedependent manner. This method is not easily scaled and is rather tedious .
Modern sequencing equipment uses the principles of the Sanger technique.
History of Sequencing
”Sanger Sequencing” developed by Fred Sanger et al in the mid 1970’s Uses dideoxynucleotides for ”chain termination”, generating fragments of different lengths ending in ddATP, ddGTP, ddCTP or ddTTP.
The 3’ hydroxyl has been changed to a hydrogen in ddNTP’s, which terminates a DNA chain because a phosphodiester bond cannot form at this 3’ location
The Sanger Technique Principle :: The Sanger Technique uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc) These are molecules that resemble normal nucleotides but lack the normal -OH group. Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops.
Requirements for Sanger Method DNA to be sequenced must be in single strand form. The region to be sequenced must be 3’ flanked by known sequence.
Reagents needed are: • A primer complementary to the known region to start and direct chain synthesis. (15-30 nucleotides in length) • DNA polymerase. • 4 deoxynucleotide triphosphates (dNTPs). • 4 dideoxynucleotide triphosphates (ddNTPs) ( small proportion ) .
Mechanism of DNA polymerization O
O -O
5’
P
-O
Base
O-
O H
O-
5’
O
O-
DNA polymerase catalyzed nucleophilic attack of the 3’-OH on a phospho-anhydride
H
P
OBase
O-
O H
H
H
H O
O
H
H
O
H
H
H O
Base O
H
H
O
P
H
P
O
H
P
OBase
O-
O H
H
O
H
H
O
H
P
O-
O-
Base
O O
O H H
H
: OH
P O-
O
P
O
O-
P
O
H
H
P
OBase
O
O O
H
H
:
-O
O
H H H
3’ O
O
Base
O
O Base
O
O H
O-
H
H
-O
P O-
O
P
OH
O-
H OH
O
H
** Since the 3’ –OH is changed to a –H in ddNTPs, it is unable to form a phosphodiester bond by nucleophilic attack on the phosphate, and it will cause a termination in the DNA chain
H
H
OH
H
H
H
3’
Sanger Method • 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
The template DNA pieces are replicated, incorporating normal nucleotides, but occasionally and at random dideoxy (DD) nucleotides are taken up. This stops replication on that piece of DNA . The result is a mix of DNA lengths, each ending with a particular labeled DDnucleotide. Because the different lengths ‘travel’ at different rates during electrophoresis, their order can be determined .
Sanger dideoxy sequencing: basic method 3’
5’
T
T T
T
5’
3’ ddA ddA ddA ddA
ddATP in the reaction: anywhere there’s a T in the template strand, occasionally a ddA will be added to the growing strand
Sequencing of DNA by the Sanger method
3’ primer 5’ ddATP GGCA
5’
CCGTAC 3’ dNTP ddTTP
ddCTP
ddGTP
GGCAT
GGC
G GG GGCATG
A T C G
3’ the sequence is complementary to the original strand
5’
*Originally four separate sets of DNA, primer and a single different DD nucleotide were produced and run on a gel. *Modern technology allows all the DNA, primers, etc to be mixed and the fluorescent labeled DDnucleotide ‘ends’ of different lengths can be ‘read’ by a laser. *Additionally, the gel slab has been replaced by polymer filled capillary tubes in modern equipment .
Gel Separation • The reaction mixtures are separated on a denaturing polyacrylamide gel. – Polyacrylamide can separate the DNA strands which differ in length by only one nucleotide. • Each band corresponds to a sequence of DNA which was terminated by a particular ddNTP. • This ddNTP is identified by lane in the radioactive method or by color in the fluorescent method. • The lowest band on the gel is the shortest. The shorter the strand, the earlier in the synthetic reaction the ddNTP was incorporated. • The lowest band on the gel is at the 5’ end of the new synthesized strand and is complementary to the 3’ end of our unknown fragment.
Visualization Methods • Two forms of labeling: – Radioactive • Primer labeled (32P or 33P) • dNTP labeled (35S or 32P)
– Nonradioactive ( Fluorescence )
*Primer labeled *ddNTP labeled - ddNTPs chemically synthesized to contain fluorescence . - Each ddNTP fluoresces at a different wavelength allowing identification .
Gel Visualization • Radioactive method which requires four gel lanes, one for each reaction vessel. – Readout is done by hand or with a densitometric scanner. • Nonradioactive fluorescence sequencing requires only one gel lane because each nucleotide has a distinct color. – The readout process is done by laser scanner and recorded by computer.
Gel Electrophoresis and Readout of Reaction Products: Nonradioactive vs. Radioactive ddGTP
ddATP
ddTTP
ddCTP
Sequence of unknown fragment
Sequence of unknown fragment
Longest synthesized band = 3’ end of synthesized strand
Shortest synthesized band = 5’ end of synthesized strand
Manual vs. Automatic Sequencing Comparison list
Manual
Automatic
DNA labeling
Radioisotope
Fluorescence dye
Signal detect
Film
Sample Read length (average)
Photomultiplier Computer 4 lanes / 1 lane / 1 sequencing sample 1 sequencing sample 250∼500 bp
400 ∼1000 bp
Fluorescent Primer Labeled Sequencing 1. Unknown fragment
6. One type of ddNTP per reaction
2. with region of known sequence
7. DNA Polymerase
3. Four separate reactions Why?
8. ddNTP incorporation stops chain synthesis
4. Fluorescent labeled primer. Different fluorescent dye per reaction
9. Run the products on gel. One or four
lanes ?
5. dNTP’s (dATP, dGTP, dCTP, and dTTP)
ddATP 5’
ddGTP 3’
3’
5’ 5’
Reaction 1
ddCTP
3’
3’
5’ 5’
Reaction 2
ddTTP 3’
3’
5’
Reaction 3
5’
3’
3’
5’
Reaction 4
Fluorescent Dideoxynucleotide Labeled Sequencing 1. Here you have one reaction vessel, with four copies of unknown fragment? 5’
2. A region of known sequence 3. Complementary primer 4. dNTP’s (dATP, dGTP, dCTP, and dTTP)
5. Fluorescent labeled ddNTP’s. Each labeled with a different fluorescent dye 6. DNA Polymerase 7. Again ddNTP incorporation stops chain synthesis 8. Run the products on gel
3’
ddGTP
3’
5’
5’
3’
3’
ddATP 5’
5’
3’ 5’
3’ 5’ 3’
ddCTP
3’
ddTTP 5’
One reaction vessel
Chemical Degradation Method •
Maxam-Gilbert method – The single stranded DNA fragment to be sequenced is labeled with radioactive phosphate at 5’ end . – The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base . – This will results in strands of varying lengths. – Strands separated out with electrophoresis. – Gels read with radioautography.
Reagents for cleaving DNA • Aliquot 1 Cleavage at only G – DNA treated with Dimethyl sulfate (DMS) – Methylation of G residues at the N7 position – the glycoside bond of the methylated G residue is hydrolyzed and the G residue is eliminated. – Piperidine is added which reacts with hydrolyzed sugar residue, cleavage of the backbone results • Aliquot 2 cleavage at G and A – Use acid instead of DMS – Position of A revealed • Aliquot 3: cleavage at C and T – Treat with hydrazine, then piperidine • Aliquot 4: cleavage at C only – Treat with hydrazine in the presence of 1.5 M NaCl – Position of T revealed • The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified
Comparison • Sanger Method – Enzymatic – Requires little if any DNA purification – No labeling of the DNA sequencing template – Requires DNA synthesis – Termination of chain elongation – Automation is available (sequencers)
• Maxam Gilbert Method – Chemical – Requires lots of purified DNA, and many intermediate purification steps – labeling of the DNA strand – Requires long stretches of DNA – Breaks DNA at different nucleotides – Relatively short readings – Automation not available (sequencers)
