Wilson.G.AnandarajI M.Sc Environmental BiotechnologyBharadhidasan University.

FOR CONTACT: anandaraj.wilson@gmail.com

WHAT IS DNA SEQUENCING ?

DNA sequencing usually involves enzymatic DNA synthesis in the presence of base-specific dideoxynucleotide chain terminators.

Determining the DNA sequence is therefore useful in basic research studying fundamental biological processes, as well as in applied fields such as diagnostic or forensic research.

FOUNDERS OF SEQUENCING TECHNOLOGY

Sanger Wally Gilbert

MAXAM & GILBERT DNA SEQUENCING (CHEMICAL DEGRADATION)

In the late 1970s, A. M. Maxam and W.Gilbert devised the first method for sequencing DNA fragments containing up to ≈500 nucleotides.

the sequence of a double-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions.

it is the early method involving base-specific chemical modification and subsequent cleavage of DNA.

most of the chemicals used in chemical degradation method are toxic and hazardous to the health of the researchers doing the DNA sequencing.

CHEMICAL DEGRADATION METHOD

PROCEDURE

four samples of an end-labeled DNA restriction fragment arechemically cleaved at different specific nucleotides.

the resulting sub-fragments are separated by agarose gel electrophoresis and the labeled fragments are detected by autoradiograph.

the sequence of the original end-labeled restriction fragment can be determined directly from parallel electrophoretograms of the four samples.

CHEMICALS INVOLVED

Dimethyl sulphate methylates guanine.

Acid removes any purines.

Hydrazine modifies any pyrimidine.

Hydrazine with NACL specifically modifies cytosines.

Piperidine is used to remove the modified bases.

SANGER METHOD

F. Sanger and his colleagues developed a second method of DNA sequencing, which now is used much more frequently than the Maxam-Gilbert method.

The sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains, these chains terminating at specific nucleotide positions.

Sanger method is the most suitable method for automation in large scale sequencing projects, and most general sequencing is now carried out in this way.

WHY SANGER METHOD TERMED AS CHAIN TERMINATION METHOD????

Specific terminators of DNA Chain Elongation is 2’3’-dideoxy nucleoside triphosphates (ddNTPs) can be incorporated normally into a growing DNA chain through their 5’triphosphate groups.

ddNTPs are telogens ,they can not form phospho - diester bonds with the next incoming deoxynucleotides (dNTPs).

Nucleotides which causes chain termination because they lack 3’ hydroxyl group for extension .hence this technique called as dideoxy (or) chain termination method.

CHAIN TERMINATION METHOD

PRINCIPLE

The single-stranded DNA to be sequenced serves as the template strand for in vitro DNA synthesis; a synthetic 5′-end-labeled oligodeoxynucleotide is used as the primer.

When a small amount of a specific dideoxy NTPs (ddNTPs) is included along with the four deoxy NTPs normally required in the reaction mixture for DNA polymerase.

The products are the series of chains that are specifically terminated at the dideoxy residue.

Thus four separate reactions, each containing a different dideoxy NTP,can be run, and their products displayed on a high-resolution Acryl amide gel.

CHAIN TERMINATION METHOD

PROCEDURE

Prepared the starting material for a chain termination sequencing experiment is a.identical single-stranded DNA molecules.

To anneal a short oligonucleotide to the same position on each molecule, this oligonucleotide subsequently acting as the primer for synthesis of a new DNA strand that is complementary to the template catalyzed by DNA polymerase requires requires the four deoxyribonucleotide triphosphates (dNTPs - dATP, dCTP, dGTP and dTTP) as substrates, would normally continue until several thousand nuceotides had been polymerised.

The polymerase does not discriminate between dNTPs and ddNTPs, so thedideoxynucleotide can be incorporated into the growing chain, but it then blocks further elongation because it lacks the 3′-hydroxyl group needed to form a connection with the nucleotide.

This process continue until several hundred nucleotides have been polymerized before a ddATP is eventually incorporated. The result is therefore a set of new chains, all of different lengths, but each ending in ddATP.

Now the polyacrylamide gel comes on to play. The family of the presence of ddNTPAs(ddATP,ddCTP,ddGTP,ddTTP) loaded into four adjacent wells of the gel.

After electrophoresis, the DNA sequence can be read directly from the positions of the bands in the gel.

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CHAIN TERMAINTAION METHOD

HOW DOES THE DNA TEMPLATE OBTAINED?

The template for a chain termination experiment is a single-stranded version of the DNA molecule to be sequenced. There are several ways in which this can be Obtained;

The DNA can be cloned in a plasmid vector.

The DNA can be cloned in a bacteriophage M13 vector.

The DNA can be cloned in a phagemid.

PCR can be used to generate single-stranded DNA.

THE DNA CAN BE CLONED IN A PLASMID VECTOR

The double stranded plasmid DNA strand must be converted into single-stranded DNA by denaturation with alkali or by boiling. This is a common method for obtaining template DNA for DNA sequencing largely.

A single strand of plasmid is that it can be difficult to prepare plasmid DNA that is not contaminated with small quantities of bacterial DNA and RNA, which can act as spurious templates or primers in the DNA sequencing experiment.

THE DNA CAN BE CLONED IN A BACTERIOPHAGE M13 VECTOR

M13 bacteriophage has a single-stranded DNA genome which,after infection of Escherichia coli bacteria, is converted into a double-stranded replicative form.

At the same time the infected cells continually secrete new M13 phage particles, approximately 1000 per generation ,these phages containing the single-stranded version of the genome.

The one disadvantage is that DNA fragments longer than about 3 kb suffer deletions and rearrangements when cloned in an M13 vector, so the system can only be used with short pieces of DNA.

PCR CAN BE USED TO GENERATE SINGLE STRANDED DNA

One way of using PCR to prepare template DNA for chain termination sequencing. The PCR is carried out with one normal primer (shown in red), and one primer that is labeled with a metallic bead (shown in brown). After PCR, the labeled strands are purified with a magnetic device.

RECENT INNOVATIONS IN CHAIN TERMINATION SEQUENCING

Thermal cycle sequencing

Automated DNA sequencing

Pyrosequencing

Sequencing by hybridization

THERMAL CYCLE SEQUENCING ( Sears et al., 1992)

The discovery of thermo stable DNA polymerases, which led to the development of PCR has also resulted in new methodologies for chain termination sequencing.

Thermal cycle sequencing has two advantages over traditional chain termination sequencing 1) uses double-stranded rather than single-stranded DNA as the starting material. 2) very little template DNA is needed, so the DNA does not have to be cloned before being sequenced.

Thermal cycle sequencing is carried out in a similar way to PCR but just one primer is used and each reaction mixture includes one of the ddNTP. Because there is only one primer, only one of the strands of the starting molecule is copied, and the product accumulates in a linear fashion, not exponentially as is the case in a real PCR.

The presence of the ddNTP in the reaction mixture causes chain termination, as in the standard methodology, and the family of resulting strands can be analyzed and the sequence read in the normal manner by polyacrylamide gel electrophoresis

THERMAL CYCLE SEQUENCING

Thermal cycle sequencing.

PCR is carried out with just one primer and with a dideoxynucleotide present in the reaction mixture. The result is a family of chain-terminated strands - the ‘A' family in the reaction shown. These strands, along with the products of the C, G and T reactions, are electrophoresed as in the standard methodology

AUTOMATED DNA SEQUENCING (Leroy Hood and colleagues,1986)

The most dramatic advance in sequencing and the one that carried DNA sequencing into a high throughput environment was the introduction of automated sequencing using fluorescence-labeled dideoxy-terminators.

In 1986, Leroy Hood and colleagues reported on a DNA sequencing method in which the radioactive labels, autoradiography, and manual base calling were all replaced by fluorescent labels, laser induced fluorescence detection, and computerized base calling.

In their method, the primer was labeled with one of four different fluorescent dyes and each was placed in a separate sequencing reaction with one of the four dideoxynucleotides plus all four deoxynucleotides.

Fluorolabeling has been equally important in the development of sequencing methodology, in particular because the detection system for fluorolabels has opened the way to automated sequence reading.

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AUTOMATED DNA SEQUENCING WITH FLUORESCENTLY LABELED DIDEOXYNUCLEOTIDES

(A) The chain termination reactions are carried out in a single tube, with each dideoxynucleotide labeled with a different fluorophore.In the automated sequencer, the bands in the electrophoresis gel move past a fluorescence detector, which identifies which dideoxynucleotide is present in each band. The information is passed to the imaging system.

(B) The printout from an automated sequencer. The sequence is represented by a series of peaks, one for each nucleotide position. In this example, a green peak is an ‘A', blue is ‘C', black is ‘G', and red is ‘T'.

PYROSEQUENCING

A novel DNA sequencing method in which addition of a nucleotide to the end of a growing polynucleotide is detected directly by conversion of the released pyrophosphate into a flash of chemiluminescence's.

Does not require electrophoresis or any other fragment separation procedure and so is more rapid than chain termination sequencing.

Template is copied in a straightforward manner without added ddNTPs.

During sequencing the addition of a nucleotide to the end of the growing strand is detectable because it is accompanied by release of a molecule of pyrophosphate, which can be converted by the enzyme sulfurylase into a flash of chemiluminescence's.

Each dNTP is therefore added separately, one after the other, with a nucleotidase enzyme also present in the reaction mixture so that if a dNTP is not incorporated into the polynucleotide then it is rapidly degraded before the next dNTP is added.

PYROSEQUENCING

The strand synthesis reaction is carried out in the absence of dideoxynucleotides. Each dNTP is added individually, along with a nucleotidase enzyme that degrades the dNTP if it is not incorporated into the strand being synthesized. Incorporation of a nucleotide is detected by a flash of chemiluminescence induced by the pyrophosphate released from the dNTP. The order in which nucleotides are added to the growing strand can therefore be followed.

SEQUENCED BY HYBRIDIZATION

The Hybridization technique is a very different approach to DNA sequencing through the use of DNA chips might one day be possible.

A chip carrying an array of different oligonucleotides could be used in DNA sequencing by applying the test molecule.

Hybridization to an individual oligonucleotide would indicate the presence of that particular oligonucleotide sequence in the test molecule, and comparison of all the oligonucleotides to which hybridization occurs would enable the sequence of the test molecule to be deduced.

The problem with this approach is that the maximum length of the molecule that can be sequenced is given by the square root of the number of oligonucleotides in the array.

SEQUENCED DY HYBRIDIZATION

The chip carries an array of every possible 8-mer oligonucleotide.

The DNA to be sequenced is labeled with a fluorescent marker and applied to the chip, and the positions of hybridizing oligonucleotides determined by confocal microscopy.

Each hybridizing oligonucleotide represents an 8-nucleotide sequence motif that is present in the probe DNA.

The sequence of the probe DNA can therefore be deduced from the overlaps between the sequences of these hybridizing oligonucleotides.

RECENT TRENDS IN DNA SEQUENCING

BIOINFORMATICS TOOLS FOR DNA SEQUENCING