DNA Sequencing

DNA sequencing used to determine the actual DNA sequence of an organism.

DNA sequencing
ACGTGACTGAGGACCGTG

CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT

DNA sequencing
DNA sequencing refers to the methods and technologies that used to determine the orders of nucleotide bases in a DNA molecule, namely adenine (A), guanine (G), cytosine (C) and thymine (T). The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of fluorescence-based sequencing methods with automated analysis, DNA sequencing has become easier and orders of magnitude faster.

DNA sequencing
DNA sequencing enables us to perform a thorough analysis of DNA because it provides us with the most basic information of all: the sequence of nucleotides. The knowledge of DNA sequences has formed the basis of basic biological researches and clinical genetic diagnosis. There are also numerous applied technology fields such as biotechnology, forensic science and biological systematics that are heavily dependent on the information generated through DNA sequencing.

DNA sequencing
DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions (i.e. clusters of genes or operons), full chromosomes or entire genomes. Depending on the methods used, sequencing may provide the order of nucleotides in DNA or RNA isolated from cells of animals, plants, bacteria, archaea, or virtually any other source of genetic information. The resulting sequences may be used by researchers in molecular biology or genetics to further scientific progress or may be used by medical personnel to make treatment decisions or aid in genetic counseling.

History
RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the complete genome of Bacteriophage MS2, identified and published by Walter Fiers in 1972 and 1976. Frederick Sanger developed rapid DNA sequencing methods with chain-terminating inhibitors" in 1977. Walter Gilbert and Allan Maxam at Harvard also developed sequencing methods, including one for "DNA sequencing by chemical degradation". In 1973, Gilbert and Maxam reported the sequence of 24 basepairs using a method known as wandering-spot analysis. Advancements in sequencing were aided by the concurrent development of recombinant DNA technology, allowing DNA samples to be isolated from sources other than viruses.

History
The first full DNA genome to be sequenced was that of bacteriophage X174 in 1977. Leroy E. Hood's and Smith announced the first semi-automated DNA sequencing machine in 1986. In 1995, Venter, Hamilton Smith, and colleagues published the first complete genome of a free-living organism, the bacterium Haemophilus influenzae. The circular chromosome contains 1,830,137 bases and its publication in the journal Science marked the first published use of whole-genome shotgun sequencing, eliminating the need for initial mapping efforts. Several new methods for DNA sequencing were developed in the mid to late 1990s. These techniques comprise the first of the "nextgeneration" sequencing methods. In 1996, Pl Nyrn and his student Mostafa Ronaghi published their method of pyrosequencing. A year later, Pascal Mayer and Laurent Farinelli describing DNA

DNA sequencing
Determination of nucleotide sequence 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

Maxam and Gilbert Method

The method developed by Maxam and Gilbert based on chemical modification of DNA and subsequent cleavage at specific bases to generate a nested set of labeled fragments. Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning. While powerful and accurate, this method requires the use of toxic chemicals. This method's use of radioactive labeling and its technical complexity discouraged extensive use after refinements in the Sanger methods had been made. This method originated in the study of DNA-protein interactions (footprinting), nucleic acid structure and epigenetic modifications to DNA, and within these it still has important applications.

Maxam and Gilbert Method

Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. Recall that the fragments in the set increase in length one base at a time from the 5 end of original labeled strand. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation.

Maxam and Gilbert Method

The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5phosphate It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5terminal The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base
1. Aliquot A + dimethyl sulphate, which methylates guanine residue 2. Aliquot B + formic acid, which modifies adenine and guanine residues 3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine

The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.

 A methyl group is added to guanine, the modified base is removed from its sugar by heating, and the exposed sugar is removed from the backbone by heating in alkali. To cleave at both A and G, the procedure is identical except that a dilute acid is added after the methylation step. The reactions that cleave at C, or at C and T, involve hydrazine to remove the bases and piperidine to cleave the backbone. The extent of the reaction can be carefully limited so that, on average, only one G is evicted from each strand, thus each strand is cleaved at only one of its guanine sites.

 A radiolabeled strand to be sequenced and the fragments created from that strand by a single cleavage at the site of G are

Each originalstrand is broken into a labeled fragment and an unlabeled fragment. All the labeled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a G along the original strand. Only the labeled fragments will be recorded once all the fragments are separated on a gel and visualized by exposing the gel to an x-ray film to create an autoradiogram of the gel.

Step 1: Preparation of Labeled Strands

Many copies of the DNA segment to be sequenced are labeled with radioisotope 32P at the 5 end of the strand. If the DNA is cloned in doublestranded form, then the 5 ends of both strands are labeled. The DNA is then denatured, copies of one strand are isolated from copies of the other strand, and each strand is sequenced separately.

Step 2: Generating a Nested Set of Labeled Fragments

Copies of one labeled strand are divided into four batches, and each batch is subjected to one of four chemical cleavage reactions. The reactions cleave the template strands at G, G and A, C, or C and T, respectively. All labeled fragments in each batch begin at the 5 end of the original strand.

Step 3: Electrophoresis and Gel Reading

The fragments from the four reactions are separated in parallel on four lanes of a gel by electrophoresis. An autoradiogram of the gel shows the positions of the labeled fragments only. Each of the four lanes is labeled by the base or bases at which the original strand was cleaved. Fragments cleaved at C show up in two lanes, the one marked C and the one marked C and T. Fragments cleaved at T are identified by noting that they appear in the lane marked C and T, but do not appear in the lane marked C.

 Fragments ending in A or G can be similarly identified. Note that the fragment cleaved at the first base will not show up on the gel, so the first base at the 5 end of the original strand cannot be determined. The band corresponding to the shortest fragments is at the bottom of the autoradiogram. The 5-to-3 sequence of the original strand is read by noting the positions and lanes of the bands from the bottom to the top of the autoradiogram.

Frederick Sanger
Discovered DNA sequencing by chain termination method Nobel Prize 1 (1958)
Complete amino acid sequence of insulin

Nobel Prize 2 (1980)

For DNA sequencing

Sanger Method
Generates the nested set of labeled fragments from a template strand by replicating the template strand to be sequenced and interrupting the replication at one of the four bases. Four different replication reactions produce fragments that terminate in A, C, G, or T, respectively. 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 dideoxynucleotides into growing strand terminates synthesis Synthesized strand sizes are determined for each dideoxynucleotide rxn by using gel or capillary electrophoresis

Dideoxynucleotide
PPP O
5 CH2 O BASE

3
no hydroxyl group at 3 end prevents strand extension

Dideoxy nucleotides
In the Sanger chain termination method, the nucleotide analog is called a dideoxynucleotide. Are added in small proportion When the correct amount is added to the solution, the chain will be terminated at each occurrence of the complementary nucleotide in the template because DNA polymerase cannot add another base to the analog. For example, if the right amount of dideoxy A is added, then the chain will be terminated at each occurrence of T in the template. To determine the complete sequence requires a separate reaction for each of the four bases A, T, C, and G. These strands are complementary to the template strand, and terminate opposite the site of a T on the template strand. Complementary strands terminating in either A, G, C, or T are produced by the inclusion in the reaction mixture of ddATP, ddGTp, ddCTP, or ddTTP, respectively.

 The primer is essential to initiate replication of the templates by DNA polymerase. The most convenient method for adding a known sequence to the 3 end of the template strand is to clone the strand in the single stranded cloning vector Ml3 so that a known M13 sequence will always flank the unknown DNA insert and can serve as the site for binding a standard primer. Also, the Ml3 cloning protocol automatically creates two types of clones, each type containing a DNA insert whose sequence is complementary to that of the other DNA insert. Thus, the two complementary strands may be sequenced and the two sequences cross-checked to ensure sequence accuracy.

DNA sequencing continued

In the dideoxy method of sequencing, the template DNA that is to be sequenced is mixed with a primer complementary to the template DNA and the four normal dNTPs, one of which is radioactively labeled for subsequent visualization purposes. This mixture is then splint into four different tubes that are labeled A, C, G, and T. Each tube is then spiked with a different ddNTP (ddATP for tube A, ddCTP for tube C, ddGTT for tube G, or ddTTP for tube T). DNA polymerase is added and using the DNA template and its complementary primer, the synthesis of new strands of DNA complementary to the template begins. Occasionally a dideoxynucleotide is added instead of the normal deoxynucleotide and synthesis of that strand is terminated at that point.

DNA sequencing continued

In the tube containing ddATP, some percentage of newly synthesized molecules will get a ddATP in each place that there is a T in the template DNA. The result is a set of new DNA molecules in tube A, each of which ends in an A. A similar type of reaction occurs in the three other tubes to result in molecules that end in C, G, and T in tubes C, G, and T respectively. After the synthesis reactions are complete, the products of the four different tubes are loaded onto four adjacent lane of a polyacrylamide gel and the different fragments are separated by size. The sequencing gel is able to resolve fragments that differ in size from each other by only one base.

DNA sequencing continued

After electrophoresis to separate the fragments by size, the fragments are visualized to exposing the gel to photographic film (Remember that one nucleotide was radioactively labeled). All fragments in lane A will end in an A, fragments in lane C will all end in a C, fragments in lane G will all end in a G, and fragments in lane T will all end in a T. The sequence of the DNA is read from the gel by starting at the bottom and reading upward.

Chain Termination

Chain Terminator Basics

Target Template-Primer TGCA ddA
Extend ddA A ddC AC ddG ACG ddT ddT

ddC

ddG Labeled Terminators

dN : ddN 100 : 1

3 primer 5

CCGTAC 5 3 dNTP
ddCTP ddGTP

ddATP ddTTP

GGCA
A

GGCAT
T C G

GGC

G GG GGCATG

All Possible Terminations

Sequence detection
To detect products of sequencing reaction Include labeled nucleotides Formerly, radioactive labels used Now, fluorescent labels used Use different fluorescent tag for each nucleotide Can run all four bases in same lane TAGCCACGTATCGAA*

TAGCCACGTATC*

TAGCCACG*

TAGCCACGT*

Sequence separation
Terminated chains need to be separated Requires one-base-pair resolution
See difference between chain of X and X+1 base pairs A T C G

Gel electrophoresis
Very thin gel High voltage Works with radioactive or fluorescent labels

Step 1: Template Preparation

Copies of the template strand are cloned in Ml 3. They are thus flanked at their 3 ends by a known sequence that will bind to a standard primer.

Step 2: Generating a Nested Set of Labeled Fragments

Copies of each template strand are divided into four batches, and each batch is used for a different replication reaction. Copies of the same standard primer and DNA polymerase is used in all four reactions, To synthesize fragments, all of which terminate at A, the dideoxy analog ddATP is added to the reaction mixture along with dATP, dGTP, dCTP, dTTP the standard primer and DNA polymerase 1. The ddATPs and one of the dNTPs are labeled with a radioactive isotope to produce radio- Iabeled strands. The figure shows a short template strand, the primer, the four reaction mixtures, and the labeled strands produced by each reaction. Note that the synthesized fragments from the four reaction mixtures compose the set of nested fragments needed to determine the order of the bases in the strand complementary to the template strand.

Step 3: Electrophoresis and Gel Reading

The fragments from the four reaction mixtures are loaded into four parallel lanes of a polyacrylamide gel and separated by length using electrophoresis. An autoradiogram of the gel is read as described in the main text to determine the order of the bases in the strand complementary to that of the template strand. Again, since the bands corresponding to the shortest fragments are at the bottom of the autoradiogram, the 5to-3 sequence of the strand complementary to the template strand is read from the bottom to the top of the autoradiogram.

Polyacrylamide Gel Electrophoresis

Separates fragments based on size

Dideoxy DNA Sequencing

Sequencing of DNA by the Sanger method

DNA Sequencing 5.17)

Shotgun Sequencing
Since only short stretches of DNA, several hundred to a thousand base pairs in length, can be obtained from a single sequencing gel, many shell sequences must be generated separately and then combined to determine the sequence of a much longer DNA fragment. Various strategies have been developed to generate these short sequences from the larger fragment. The shotgun approach is the most widely used in the larger sequencing projects.

Shotgun Sequencing
Copies of a long fragment to be sequenced are broken into much shorter fragments that overlap one another, and the short fragments are cloned. Those clones are then picked at random and sequenced. The sequence of the long fragment is determined by finding overlaps among the short sequences and assembling those sequences into the most likely order. Numerous computer algorithms have been developed to facilitate the assembly of long sequences.

Shotgun Sequencing
Inevitably, gaps remain in the sequence of the long fragment, and they are filled by switching to a directed sequencing strategy. That is, the short clones are no longer sequenced at random, but rather, short sequences at the end of a continuous stretch of known sequence provide the information necessary to construct a probe to pick out a clone, or region of a clone, whose sequence will extend the known sequence. Most of the large sequencing projects to date have used a mixture of random and directed sequencing strategies to complete the sequence of long, contiguous stretches of DNA. The advantage of the random, or shotgun, strategy is that in the course of picking clones at random and sequencing them, any given region is usually sequenced many times, thereby reducing the errors in the final sequence.

Automated DNA sequencing

In automated DNA sequencing a radioactive deoxynucleotide is not used and all four dideoxy reactions are done in a single tube. This is possible because each ddNTPs is labeled with a different flourescent dye. Therefore the dye present in each synthesized fragment corresponds to the dye attached to the dideoxynucleotide that was added to terminate the synthesis of that particular fragment. The contents of the single tube reaction are loaded onto a single lane of a gel and electrophoresis is done. A flourimeter and computer are hooked up to the gel and they detect and record the dye attached to the fragments as they come off the gel. The sequence is determined by the order of the dyes coming off the gel.

Automated DNA sequencing