DNA SEQUENCING

DNA SEQUENCING

23.      DNA SEQUENCING
It is the method of determining the order of the nucleotides adenine, guanine, cytosine and thymine in a molecule of DNA.
23.1.    Sanger Sequencing-
This technique was discovered by Fredrick Sanger. The basic principle of this technique is the use of dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators.


These ddNTPs works as chain terminators because they don’t have –OH group on its 3’ carbon for the elongation of the nucleotide chain. 
Material required - single-stranded DNA template + DNA primer + DNA polymerase +  radiolabeled nucleotides + ddNTPs
23.1.1.    Process
Sample divided into four subsamples which contains all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and containing DNA polymerase. 
To each subsample one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). 
Now DNA fragments are heat denatured and resolved by gel electrophoresis on a denaturing polyacrylamide-urea gel. Different bands on gel electrophoresis are obtained due to the differential polymerization of DNA chains because incorporation of ddNTPs halts further elongation of daughter strands.
23.1.2.    Dye-terminator sequencing 
It is the advanced version of chain termination sequencing in which ddNTPs are labelled with different fluorescent molecules and each molecule have different wavelengths of fluorescence and emission. So this method provides direct determination of sequence in a single tube only.
Due to its high reliability and fast speed, it is used over the chain terminator sequencing.
23.1.3.    Application
Azidothymidine (AZT) drug used for the treatment of AIDS. AZT (also called as zidovudine) is an analogue of thymidine and readily taken up by cells in its triphosphate form. The reverse transcription of the human immunodeficiency virus (HIV) halts because AZT has no 3′ -OH group.
23.1.4.    Sanger method is based upon enzymatic reaction while the Maxam & Gilbert method based upon chemical reaction so the Sanger method is 2 to 3 time faster.


23.1.5.    It is cheaper because no chemical is used.
23.1.6.  The DNA sequence is complementary to the observed data.
23.2.    Maxam Gilbert Sequencing
Discovered by Allan Maxam and Walter Gilbert
This DNA sequencing method based on the chemical modification of DNA sequence and then cleave at specific bases.
The sample of DNA is divided into four proportions and each sample is radiolabeled at 5’ ends then it is treated with different Chemical and generates changes in it. For example, the purines (A+G) are depurinated using formic acid, the guanines are methylated by dimethyl sulfate, and the pyrimidines (C+T) are methylated using hydrazine.
Now the modified base is cleaved by use of hot piperidine. Now the sample is electrophoresis to resolve all DNA molecules and to visualize the fragments, the gel is exposed to X-ray film for autoradiography. So the position of all nucleotide bases in a sequence is identified.
23.2.1.    The application


The sequence of the individual gene
Metagenomics–Identification of species present in a water body, sewage, dirt.
Analysis of protein structure and function sequence
Possible for any disease. 
Detection of mutation
Kinship study. 
DNA fingerprinting
23.2.2.    Radiolabelling of DNA is done by kinase using \gamma p32 ATP.


DMS modifies the N7 of Guanine.
DMS & Hydrazine breaks the Glycosidic bond between base and sugar.
Hot piperidine breaks phosphodiester bond at 3' carbon.
1.5 M NaCl Prevent Thymidine reaction
DMS + Piperidine occur only G reaction.
DMS + Piperidine + formic Acid occur (A + G) reaction.
Data is read from bottom to top process of base calling would involve.
Advantage & application.
First-time exon Intron theory proposed and find out that intron was flanked by the same dinucleotide at 5' GT and 3' AG and proposed eukaryotic and prokaryotic coding gene same in the organisation.
It is applicable only 200 - 300 long bp DNA fragment.     
23.3.    Next-gen sequencing-
These technologies are basically needed of the time with respect to lower the cost of reactions, make techniques cheaper, faster and more reliable and accurate results. 
23.3.1.    Pyrosequencing / Roche / 454 Sequencing
The main disadvantage of dideoxy sequencing is that it requires gel electrophoresis and to fractionate newly synthesized DNA fragments. This technique is able to detect the sequence of the new polynucleotide chain with the reactivity of polymerase. Roche 454 was the first successful pyrosequencer used.


Polynucleotide chains synthesized by polymerase incorporates dNMP cleaving alpha and beta ester bond and releases PPi in the solution. Now the enzyme reactions proceed which detects PPi and ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). Now formed ATP is utilized by bioluminescence molecule luciferin and converts to oxyluciferin in the presence of luciferase and generates visible light in amounts that are proportional to the amount of dNTP utilized which is recorded by a CCD camera. 
Any unused dNTPs and excess ATP which are not incorporated in polynucleotide chain are degraded by the enzyme apyrase, which is included in the reaction mixture. 
23.3.2.    Single molecule real time sequencing (SMRT)
This technique is based on the sequencing by synthesis approach in which sequencing is along with the synthesis of the new strand. This technique was first developed by Pacific Biosciences. It is 20,000 times faster than the second generation sequencers.


The DNA is synthesized in scale zero-mode waveguides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well.
The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. SMTR technology allows the detection of nucleotide modifications.
This technique uses two unique features which are-
First, use of unconventional fluorophore-labelled dNTPs, where normally flurophore labelled dNTPs incorporate into nucleotide chain and then halts polymerization after few nucleotides because of stearic hinderence; these dNTPs are labeled terminally so that new chains have no problem of the hindrance.
Second is the SMRT chip which is a very thin metal film affixed on a glass substrate and this metal film contains thousands of tiny sub-wavelength holes known as nanophotonic zero-mode waveguides (ZMWs). An ZMW has a volume of just 20 zeptoliters (l0-2l liters) and these nanopores have immobilized single stranded DNA and polymerase enzyme in it.


Now differntialy fluorophore labeled dNTPs move freely in the solution over pores and the excitation illumination is directed towards the bottom of pores. When a dNTP binds with its complementary base an peak increase in intensity is observed and as the fluorochrome is released intensity falls down. This is how the sequence is identified.
23.3.3.    SOLiD (Sequencing by Oligonucleotide Ligation Detection)
This technology was purchased by Life Technologies / Applied Biosystems Inc. in 2006. The sequencer adopts the technology of two-base sequencing based on ligation sequencing. This technique uses an emulsion-based PCR technique for the amplification of individual DNA fragments. These ligation probes are 8-mer probes.
These probes have free 3’ –OH group and a fluorescent dye at the 5’ end and a cleavage site between the fifth and sixth nucleotide. Now, these probes are hybridized with the target sequence and ligated region are sequenced. 
23.3.4.    Illumina (Solexa) sequencing 
This technique was developed by S Balasubramanian and D Klenerman of Cambridge University and founded Solexa company. This sequencing technology uses reversible dye terminators for the sequencing of complex regions of a genome and highly repetitive sequences. 
In this technique, four types of terminator dyes ligated with different fluorochromes are used and they starts to compete with binding with their complementary sites.
DNA molecule are bridge amplified and then terminator dyes are added to it which binds one by one to complementary base. Laser light is used to remove the terminator group from the 3’ end and sequencing was done. This technique shorter sequences but it is faster and cheaper than other techniques. 
23.3.5.    Polony sequencing 
This techniques was first developed by Dr. George Church in Harvard Medical college. It is an inexpensive, accurate, high-throughput technique to sequence genomes of interest by comparing it with a reference genome. It uses an in vitro paired-tag library with emulsion PCR, an epifluorescence microscope, and computer-controlled flowcell/fluidics system.
23.3.6.    ION Torrent sequencing
It is similar to 454 pyro sequencing technology but it does not use fluoroscent labled nucleotides.
It is based on th detection of the hydrogen ION released during the sequencing process.
ION torrent uses a chip that contains a set of micro wells and each has a bead with several identical fragments the incorporation of each Nucleotide a Hydrogen ION is released which change the pH of the solution this change is detected by a sensor attached to the bottom of the micro well and converted into a voltage signal which is proportional to the number of nucleotide incorporated.
Disadvantage
Difficuilty of interpreting the homopolymer sequence (more than 6 bp) which cause insertion and deletion.
error approximately 1%.
23.3.7.    Automated fluoroscence sequencing
Fluorscence labeled dideoxy terminators is used in AFS.
Radioactive lables, autoradiography and manual base calling were all replaced by fluorosuence lables.
Laser induced fluoroscence detection and comperterized base calling the primer was labled with one of four different fluoroscent dyes and each was placed in separate sequencing reaction with one of the dideoxy nucleotides plus all four deoxy nucleotides.
Once the reaction were complete the four reaction were pooled and run together in one lane of a poly acrylamide gel.
A four colour laser induced fluoroscence detector scanned the gel as the reaction fragment migrated past.
Fluoroscence signature of each fragment was then sent to a computer where the software was trained to perform base calling.
ddG    \Rightarrow    SF505
     A    \Rightarrow    SF512
     C    \Rightarrow    SF519
   T    \Rightarrow    SF526        These are dyes of a dideoxy nucleotide
23.4.    Whole Genome Sequencing :
1.    Shotgun sequencing
Shotgun sequencing is the technique to sequence the whole genome like the human genome. In this huge DNA sequence is fragmented into the smaller fragments less than 300 bp each randomly by the sonication. After the formation of the smaller fragments. Each fragment is sequenced individualy with the help of any DNA sequencing method like sanger-chain termination method, automatecl sanger sequencing after the sequencing of all the smaller fragments etc. After the sequencing of all the smaller fragments. the data is compiled by the computer software, the computer assemble all the fragments to find out ovelapping regions as the whole genome was broken randomly into smaller fragments of different length, so there are the number of overlapping regions and through these overlapping regions and through these overlapping regions the whole genome is sequenced. 

2.    Primer walking/chromosome walking :
Primer walking is a sequencing method for the large DNa fragments of about 1.3 - 7 kb. These large fragments are very difficult to be sequenced in a single sequence read using the sanger chain termination method. Primer walking involves the following steps :
1.    Primer of known DNA sequence added at the end of the DNA to be sequenced. The new short DNA sequence of about 300 bp is sequenced used the chain termaination method. 
2.    Now the end of the sequence of DNA which is sequenced ley the chain termination method will serve as the primer for the next part of the DNA to be sequenced. Again by the chain termination method, the part of the long DNA sequence is sequenced and again the end part of this will seume as perimer for the next part of the long DNA sequence.
As fecom, the alcove steps of eprimer walking it seems that primer is walking on DNA hence the term is used for the sequencing is primer walking.

Clone-cantig technique :
In this method of sequencing, the long DNA is sequenced are fragmented by the specific restriction endonucleases which cut the DNA at the specific site different RE are to create overfeapping fragments. 
The fragments formed after the RE digestion will be ligated in the vector and the vector used can be cosmid, BAC etc. After the ligation, vectors are incorfoorated in the bacterial cells via Transformation and these bacterial cells are allowed to grow in the culture medium. After the sufficient growth in the medium, the bacterial cells are isolated and then the DNA segments incorporated via transformation of vector also isolated and these fragments now undergo sanger method of sequencing. The set of overlapping sequences are obtained after the Sanger method of sequencing and these overlapping sequence of DNA fragments is known as conting. These contig will assemble and the sequence of long DNA can be sequenced by the overlapping fragments.
Cloning is done to increase the quantity of sample and to get the overlapping fragments in a huge amount.


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