Sequencing

 Determination of nucleotide sequence in DNA is the important technique in molecular

biology.

 This technique was first developed by the two scientist groups in 1975.

 Maxam AM and Gilbert W. American scientists at Havard University developed the

nucleotide sequencing technique using the chemical method.

 Sanger F, Niklen S and Coulson AR, the Bristish scientists at Cambridge successfully

developed this technique using the enzymatic method.

 Nowadays these techniques are widely used in molecular biology research.

 The strategies for DNA sequencing consists of 4 steps:

1. Preparation of DNA template

2. Sequencing reaction

3. Electrophoresis

4. Analysis

1. Preparation of DNA template

The DNA template used for sequencing can be prepared by several methods:

a) Gene cloning
 It is conventional method.

 Isolate and purify genomic DNA.

 Cut with appropriate restriction enzyme.

 Cut sequencing vector with the same enzyme.

 Insert DNA fragment into vector.

 Transfer into bacterial host cell.

 Select host cell containing recombinant DNA.

 Isolate and purify recombinant DNA and used as a DNA template for nucleotide sequencing.

b) DNA amplification by PCR

DNA fragment to be sequenced can be produced from one or two copies to million.

c) The Lambda exonuclease treatment method

It is a method to produce single stranded DNA template after PCR. By this method, the DNA region to be sequenced is amplified by PCR with a pair of primers. The 5’ end of one primer is phosphorylated using T4 polynucleotide kinase enzyme whereas another primer is not. After PCR, a strand of PCR product should consist of 5’ phosphate group whereas another strand consists of 5’

hydroxyl group. Then these products are treated with lambda exonuclease enzyme which can digest a DNA strand starting from 5’ phosphate group. Another strand containing a hydroxyl group at 5’ end is resistant to this enzyme activity. This final product of this treatment is a single stranded DNA which consists of 5’ hydroxyl

group and can be used as DNA template for sequencing.

d) Assymetric PCR

It is a technique used for amplification and production of a single strand DNA. In this method, the DNA fragment is amplified by using a couple of primer which contain different concentration (50:1 or 100:1 mole). The single stranded DNA fragment will be produced from a primer that has a highest amount in the reaction after 30 cycles if PCR and can be used as a template for nucleotide sequencing.

e) Nowadays, the double stranded DNA fragment produced by PCR can be used as template directly for the sequencing method called thermal cycle sequencing. This method has advantages over the other methods in terms of sensitivity because this method can sequence DNA template as low as 50 mol and the protocol is simple.

2. Sequencing reaction

The DNA templates are divided into 4 reactions for sequencing protocols. This reaction can either be performed in the chemical method of Maxam and Gilbert or an enzymatic method of Sanger. They depend on the production of a mixture of oligonucleotides labelled either radioactivity 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 polyacrylamide gels and the position of the bands determined.

THE MAXAM AND GILBERT TECHNIQUE

Principle

Chemical degradation of purines

1) Purines (A and G) damaged by dimethylsulfate

2) Methylation of Base

3) Heat releases base

4) Alkali cleaves G.

5) Dilute acid cleave A>G.

Figure 1. THE MAXAM AND GILBERT TECHNIQUE

Chemical degradation of pyrimidine

1) Pyrimidines (C and T) are damaged by hydrazine.

2) Piperdine cleaves the backbone.

3) 2M NaCl inhibits the reaction with T.

Maxam and Gilbert Method

 Chemical degradation of purified fragments(chemical degradation)

 The single stranded DNA fragments to be sequenced is labelled by treatmentwith alkaline phosphatase to remove the 5’ phosphate.

 It is then followed by reaction with p-labelled ATP in the presence of polynucleotide whose which attaches P-labelled to the 5’ terminal.

 The labelled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base.

1. Aliquots A + dimethyl sulphate which methylates guanine residue.

2. Aliquots B + formic acid which modifies adenine and guanine residues.

3. Aliquotes C+ Hydrazine which modifies thymine+cytosine residues.

4. Aliquots 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.

 The concentrations of the modifying chemicals verses controlled to introduce on average one modification per DNA molecule.Therefore, a series of labelled fragment is generated.

 Cleavage produces are separated by polyacrylamide 7M urea gel

electrophoresis at high voltage. DNA fragments move in the gel according to their sizes and are detected by autoradiography. The nucleotide sequences are read from 5’ and at the bottom of the gel to the 3’ end at the top of the gel.

Advantages/ disadvantages of Maxam Gilbert Sequencing

 Requires lots of purified DNA and many intermediate purification steps.

 Relatively short readings.

 Automation non available (Sequencers)

 Remaining use for ‘foot printing’ partial protection against DNA modification when proteins bind to specific regions and that produce ‘holes’in the sequences ladder.

Original Sanger Method

 Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA.

 Requires DNA polymerase I.

 Requires a cloning vector with initial primer (high yield bacteriophage, modified by adding beta galactosidase screening, polylinkers).

 Uses 32 P-deoxynucleoside triphosphates.

Sanger Method

 In vitro DNA synthesis using ‘terminators’ use of dideoxynucleotides that do not permit

chain elongation after their integration.

 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 dideoxynucleotide into growing strand terminator synthesis.

 Enzymatic methods.

 Synthesized strand sizes are determined for each dideoxynucleotide by using gel or capillary electrophoresis.

Figure 2. Sanger Sequencing (Reprinted from "Sanger Sequencing", by BioRender, April 2020, retrieved from https://app.biorender.com/biorender-templates/t-5ef132f6c7bcd500b388a9c3-sanger-sequencing Copyright 2020 by BioRender.)

No hydroxyl group at 3’ end prevents strand extension.

The principles are:

 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

GGCATG from downward in agarose gel electrophoresis

3. Electrophoresis

Based on their sizes, the products from each sequencing reaction are separated by loading into polyacrylamide gel containing 7 M urea and electrophoresis at high voltage.

4. Analysis

After electrophoresis, the DNA fragments migrating into the gel are detected by an appropriate method which depends on the type of reporter molecules that are labelled during the sequencing reaction:

a) In autoradiography technique for the detection of the fragments consisting of radioisotope molecules such as 33P, 32P or 35S.

b) Colour detection for the labelled fragments with biotin.

c) Silver staining for the direct detection of DNA fragments in the gel.

d) The laser detection for the detection of sequencing products containing fluorescent molecules. This method is used in the automatic DNA sequencing machine.

Sequencing, in the context of molecular biology and genetics, refers to the process of determining the precise order of nucleotides (adenine, thymine, guanine, and cytosine) within a DNA or RNA molecule. It has revolutionized many fields of science and medicine, allowing researchers to understand genetic variations, study evolutionary relationships, diagnose diseases, and develop personalized medicine. Here's a detailed overview of sequencing, including its history, types, mechanism, recent research, and clinical significance:

History of Sequencing:

• 1950s-1970s: Early sequencing techniques, such as paper chromatography and gel electrophoresis, provided insights into the composition of nucleic acids but were limited in their ability to sequence entire genomes.
• 1977: Frederick Sanger and colleagues developed the first method for DNA sequencing, known as the Sanger sequencing method or dideoxy sequencing, which revolutionized the field of molecular biology.
• 1980s-1990s: Automated Sanger sequencing instruments were developed, leading to the completion of the Human Genome Project in 2003, which sequenced the entire human genome.

Types of Sequencing:

1. Sanger Sequencing: Based on the incorporation of chain-terminating dideoxynucleotides during DNA synthesis.
2. Next-Generation Sequencing (NGS): High-throughput methods that enable the simultaneous sequencing of millions of DNA fragments. Examples include Illumina sequencing, Ion Torrent sequencing, and PacBio sequencing.
3. Third-Generation Sequencing: Single-molecule sequencing technologies that provide long-read sequencing capabilities. Examples include Pacific Biosciences (PacBio) and Oxford Nanopore sequencing.

Mechanism of Sequencing:

• Sanger Sequencing: Involves DNA synthesis using DNA polymerase, dideoxynucleotides (lacking a 3' hydroxyl group), and fluorescently labeled dNTPs. Fragments are separated by size using capillary electrophoresis.
• Next-Generation Sequencing: Involves library preparation, sequencing by synthesis (e.g., Illumina), sequencing by ligation (e.g., Ion Torrent), or nanopore-based sequencing (e.g., Oxford Nanopore), followed by bioinformatics analysis to reconstruct the sequence.

Recent Research and Advances:

• Long-Read Sequencing: Third-generation sequencing technologies, such as PacBio and Oxford Nanopore, offer the ability to sequence longer DNA fragments, enabling the detection of structural variations and complex genomic rearrangements.
• Single-Cell Sequencing: Techniques such as single-cell RNA sequencing (scRNA-seq) and single-cell DNA sequencing allow researchers to analyze the transcriptome and genome of individual cells, providing insights into cellular heterogeneity and cell-to-cell variability.
• Metagenomic Sequencing: Enables the study of microbial communities in various environments, such as the human gut microbiome, soil microbiome, and ocean microbiome.

Clinical Significance:

• Diagnosis of Genetic Disorders: Sequencing technologies are used for the diagnosis of inherited diseases, cancer mutations, and rare genetic disorders.
• Pharmacogenomics: Genetic sequencing is utilized to predict an individual's response to drugs and optimize drug therapy (personalized medicine).
• Cancer Genomics: Sequencing of cancer genomes helps identify driver mutations, characterize tumor heterogeneity, and guide targeted therapies.
• Infectious Disease Surveillance: Whole-genome sequencing of pathogens facilitates the surveillance of infectious diseases, outbreak investigations, and monitoring of antimicrobial resistance.

In conclusion, sequencing has transformed our understanding of genetics, biology, and medicine. Advances in sequencing technologies continue to drive innovation in research, diagnostics, and therapeutics, with profound implications for human health and disease.