DNA Sequencing: Introduction, Types, Principle, Procedure and Uses

Introduction of DNA Sequencing

DNA sequencing is a technique used to determine the precise order of the four nucleotide bases, adenine, guanine, cytosine, and thymine which make up a strand of DNA. These bases provide the underlying genetic basis (the genotype) for telling a cell what to do, where to go, and what kind of cell to become (the phenotype). Nucleotides are not the only determinants of phenotypes, but also they are essential to their formation. Each organism has a specific nucleotide base sequence.

Sequencing an entire genome (all of an organism’s DNA) remains a complex job. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long “consensus.” e.g. Sample Comparison of the DNA sequences of a nucleoprotein gene in infections of two patients with different strains of rabies are as follows-

A. Gene sequence AY138566; rabies virus isolate 1360, India
B. Gene sequence AY138567; rabies virus isolate 945, Kenya

Line 1a gaaaaagaac ttcaagaata tgagacggca
Line 1b gagaaagaac ttcaagaata cgagacggct

Line 2a gaattgacaa agactgacgt agcgctggca
Line 2b gaactgacaa agactgacgt ggcattggca

Line 3a gatgatggaa ctgtcaattc ggatgacgag
Line 3b gatgatggaa ctgtcaactc tgacgatgag

Deoxyribonucleic acid (DNA)

DNA is a nucleic acid that functions include-

Storage of genetic information
Self-duplication and inheritance
Expression of the genetic message
DNA’s major function is to code for proteins.
Information is encoded in the order of the nitrogenous bases.

Watson and Crick model of DNA

DNA is composed of 2 chains of nucleotides that form a double helix shape.
The two strands are antiparallel.
The backbone of the DNA molecule is composed of alternating phosphate groups and sugars.
The complementary nitrogenous bases form hydrogen bonds between the strands.
A is complementary to T and G is complementary to C.

History of DNA to DNA Sequencing

1953 – the structure of DNA was established as a double helix.
RNA sequencing was one of the earliest forms of nucleotide sequencing done by Ray Wu, a Chinese American biologist based at Cornell University, who published one of the first methods for sequencing DNA in 1970.
1970 – first method of DNA sequencing involved a location
specific primer extension strategy.
1977 – Frederick Sanger published a method for DNA
sequencing with chain-terminating inhibitors.
1977 – Allan Maxam and Walter Gilbert developed
DNA sequencing by chemical degradation.
1977 – the first genome to be sequenced was that of bacteriophage φX174.
1990 – several new methods are developed in the mid to late ’90s.
2003 – Complete Human Genome Project
October 2019 -NHS introduced a new fast-track DNA test to scan for rare diseases in babies and children in South West Genomic Laboratory Hub

Purposes of DNA Sequencing

Deciphering “code of life”
Detecting mutations
Typing microorganisms
Identifying human haplotypes
Designating polymorphisms

Methods of DNA sequencing

Basic methods

Maxam-Gilbert sequencing
Chain termination (Sanger’s method) method

Advanced methods

Short Gun Sequencing
Bridge PCR Sequencing

Next-generation methods

Massively parallel signature sequencing
Polony sequence
Pyrosequencing
Illumina sequencing
Solid sequencing
DNA nano ball sequencing.

Maxam-Gilbert Method of Sequencing

A. M. Maxam and W. Gilbert-1977
The sequence of a double-stranded or single-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions.

Principle of Maxam-Gilbert Method of Sequencing

Chemical degradation
Reaction in two stages:
Chemical modification of the bases
The modified base is removed from its sugar, piperidine cleaves phosphodiester bonds 5’ and 3’, and the base is released

10 nucleotide DNA sequence: 5’P-TTCAGCCGAT-OH3’

First step: 5’P-TTCAGCCGAT-OH3’+H2O→ 5’OH-TTCAGCCGAT-OH3’+Pi 5’OH-TTCAGCCGAT-OH3’+A-P-P-32P→5’32P-TTCAGCCGAT-OH3’+ADP
[gamma-32P]ATP
The DNA solution is divided into four aliquots: G only, G+A, C+T, and C only.
Next steps: The four differently fragmented samples of DNA is simultaneously electrophoresed in parallel lanes on a sequencing gel.
After electrophoresis gel is exposed to a photographic film
The sequence of DNA simply read off this autoradiogram

Sanger’s method of DNA Sequencing

The most common approach is used to sequence DNA.
Invented by Frederick Sanger – 1977
Twice Nobel Prize winner.
Also termed as chain termination or dideoxy method
This method uses dideoxynucleotide triphosphates (ddNTPs) chain terminators: which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs). Therefore in a synthesis reaction, if a dideoxynucleotide is added instead of the normal deoxynucleotide, the synthesis stops at that point because the 3’OH is necessary for the addition of the next nucleotide is absent.

Principle

The sequence of a single-stranded DNA molecule is determined by the enzymatic synthesis of complementary polynucleotide chains. These chains terminating at specific nucleotide positions. Separation by gel electrophoresis and read DNA sequence.

Requirements

DNA sequencing is performed in four separate tubes, each containing

Single-stranded DNA to be sequenced
DNA polymerase
Primers
The four dNTPs (dATP, dCTP, dTTP and dGTP)
Small amount of one of the four ddNTPs (ddATP or ddCTP or ddTTP or ddGTP)
Either the primers or the dNTPs are radiolabeled with 32P

Getting DNA Template

The DNA can be cloned in a plasmid vector.
The DNA can be cloned in a bacteriophage M13 vector.
PCR can be used to generate single-stranded DNA.

Test Procedure Sanger’s Sequencing

The Sanger sequencing method completes in 6 steps:

The double-stranded DNA (dsDNA) is denatured into two single-stranded DNA (ss DNA).
A primer that corresponds to one end of the sequence is attached.
Four polymerase solutions with four types of dNTPs but only one type of ddNTP are added.
The DNA synthesis reaction initiates and the chain extends until a termination nucleotide is randomly incorporated.
The resulting DNA fragments are denatured into ssDNA.
The denatured fragments are separated by gel electrophoresis and the sequence is determined.

Uses of DNA Sequencing

Forensics

Identify individuals
Determine the paternity of a child
Identifies endangered and protected species

Medicine

Detect genes that are hereditary or cause diseases

Agriculture

Map the genome of microorganisms

Future of DNA Sequencing

Projects might focus on researching:

The links to develop a lifestyle
Genomic and cardiovascular disease
Early detection of cancer

Next-Generation Technologies

The most recent set of DNA sequencing technologies are collectively referred to as next-generation sequencing and they are-

Solexa: Based on whole-genome sequencing
SOLiD (Sequencing by Oligonucleotide Ligation and Detection): Ligation and detection developed by Life Technologies and has been commercially available since 2006
454 Pyrosequencing It is a method of high throughput DNA sequencing that utilizes a single strand of DNA with a length of 400-500 bp.
Helicos Single-molecule sequencing

Features of next-generation sequencing

Highly parallel: many sequencing reactions take place at the same time
Microscale: reactions are tiny and many can be done at once on a chip
Fast: because reactions are done in parallel, results are ready much faster
Low-cost: sequencing a genome is cheaper than with Sanger sequencing
Shorter length: reads typically range from 505050 -700700700 nucleotides in length

Pyrosequencing

DNA Sequencing based on the “SEQUENCING BY SYNTHESIS”
It relies on the detection of PYROPHOSPHATE release on NUCLEOTIDE incorporation, rather than CHAIN TERMINATION.
The single-strand DNA template is hybridized to a sequencing primer and incubated with the enzymes.
The pyrosequencing method is based on detecting the activity of DNA polymerase with another chemiluminescent enzyme-like-

DNA polymerase
ATP sulfurylase
Luciferase and apyrase
Substrates adenosine 5´ phosphosulfate (APS) and luciferin.

Shotgun sequencing

Shotgun sequencing, also known as shotgun cloning, is a method used for sequencing long DNA strands or the whole genome.
In shotgun sequencing, DNA is broken up randomly into numerous small segments and overlapping regions are identified between all the individual sequences that are generated.
Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing.
Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence.
The shotgun approach was first used successfully with the bacterium H. influenzae.
Craig Venter used this method to map the human genome project in 2001.

Key Notes

DNA sequencing is the process of determining the sequence of nucleotides or the order of bases (adenine, guanine, cytosine, and thymine) in a section of DNA.
Differences between Sanger’s sequencing and Maxam-Gilbert are as follows-
Sanger Maxam-Gilbert
Enzymatic Chemical
Requires DNA synthesis Requires DNA
Termination of chain Breaks DNA at different nucleotides
elongation
Automation is not available
S-S DNA ds-or ss DNA
Common four key steps of DNA sequencing are – In the first step DNA is removed from the cell. This can be achieved either mechanically or chemically. The second phase involves breaking up the DNA and inserting its pieces into vectors, cells that indefinitely self-replicate, for cloning. In the third phase, the DNA clones are placed with a dye-labeled primer (a short stretch of DNA that promotes replication) into a thermal cycler (PCR machine), a machine that automatically raises and lowers the temperature to catalyze replication. The final phase consists of electrophoresis, whereby the DNA segments are placed in a gel and subjected to an electrical current that moves them. Originally the gel was placed on a slab, but nowadays it is inserted into a very thin glass tube known as a capillary. When subjected to an electrical current the smaller nucleotides in the DNA move faster than the larger ones. Electrophoresis thus helps sort out the DNA fragments by their size. The different nucleotide bases (adenine, guanine, cytosine, and thymine) in the DNA fragments are identified by their dyes which are activated when they pass through a laser beam. All the information is fed into a computer and the DNA sequence is displayed on a screen for analysis.

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