Sequencing types for class 12th biotechnology

Content of the notes: It is accompanied with following topics:

• DNA sequencing /Genome sequencing
• Methods of sequencing (DNA/GENOME)
• Expressed sequence tags(EST)
• Sequence tag-sites(STS)

Some techniques associated with the same:

1. SAGE
2. DNA Microarray

Basic terminology:

1. DNA: DNA is the molecule that is the hereditary material in all living cells. Genes are made of DNA, and so is the genome itself. A gene consists of enough DNA to code for one protein, and a genome is simply the sum total of an organism's DNA .It is long and skinny, capable of contorting like a circus performer when it winds into chromosomes. It's skinny as a whip and smart as one too, containing all the information necessary to build a living organism.
2. Gene: A gene is a small piece of the genome. It's the genetic equivalent of the atom: As an atom is the fundamental unit of matter, a gene is the fundamental unit of heredity. Genes are found on chromosomes and are made of DNA.
3. Chromosomes: A chromosome is a package containing a chunk of a genome—that is, it contains some of an organism's genes. The important word here is "package" chromosomes help a cell to keep a large amount of genetic information neat, organized, and compact. Chromosomes are made of DNA and protein.
4. Genomes: A genome is all of a living thing's genetic material. It is the entire set of hereditary instructions for building, running, and maintaining an organism, and passing life on to the next generation. In most living things, the genome is made of a chemical called DNA. The genome contains genes, which are packaged in chromosomes and affect specific characteristics of the organism.

Further we can say that a genome is divided into chromosomes, chromosomes contain genes, and genes are made of DNA. Each species on this earth have their own distinct genes.

The information or makeup proceeds as:

DNA                         GENE                        CHROMOSOMES                        GENOMES 

Now, we will discuss the sequencing of DNA and then genome sequencing. In general term sequencing means the arrangement of particular elements in a given order. The same is the case with DNA and Genes. To understand the concept of genome sequencing we have to understand the basic principle behind it i.e, the from DNA sequencing to GENE sequencing.

1. DNA sequencing: The term DNA sequencing encompass the biochemical methods for determining the order of the nucleotides bases, adenine, guanine, cytosine and thymine in a DNA strand. Thus, we can say that It includes any method or technology that is used to determine the order of these four bases
2. The first outbreak in this field takes place when (Fred Sanger) had completed the sequence of all amino acids in the insulin(1955),then Rna sequencing is developed 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. The major contribution towards sequencing is given by Sanger who developed the chain termination method and then Maxam and Gilbert they purposed the chemical degradation method. 

The method of DNA/Genome sequencing is divided into three categories:

1. Basic methods: It includes chain termination method and the chemical degradation method.
2. Advance method: Shotgun approach and clone contig approach (Genome sequencing method )
3. Next generation sequencing or high through put methods: It includes , iilumina sequencing, soliD sequencing, nanopore sequencing etc (Genome sequencing method).

These above methods are the bases for the genome sequencing as first of all we have to extract the Dna sequence and after that genome is to be sequenced let us discuss the whole methods in details .

1. Chain termination method (Sanger): In this method the sequence of ssDNA molecule is determined by the enzymatic synthesis of complementary polynucleotide chains, these chains terminating at specific nucleotide positions. The method relies on the use of single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleosidetriphosphates (dNTPs) that lack 3’OH group, and modified di-deoxynucleotidetriphosphates (ddNTPs).

Primer:  A primer is a short strand of RNA or DNA (generally about 18-22 bases) that serves as a starting point for DNA synthesis.

DNA polymerase: DNA polymerases are enzymes that synthesize DNA molecules from deoxyribonucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule

                      ddNTPS:  Dideoxynucleotides are chain-elongating inhibitors of DNA polymerase.

In this method first the purified DNA is synthesized invitro. The target DNA is denatured and annealed to anoligonucleotide primer, which is then extended by DNA polymerase using a mixture of deoxynucleotide triphosphates (normal dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs). ddNTPs lack the 3’ OH group to which the next dNTP of the growing DNA chain is added. Without the 3’ OH, no more nucleotides can be added, and DNA polymerase falls off. The resulting newly synthesized DNA chains will be a mixture of lengths, depending on how long the chain was when a ddNTP was randomly integrated. Then split the sample into four aliquots.

When a DNA polymerase (e.g. Klenow fragment) is added to the tubes, the synthetic reactionproceeds until, by chance, a dideoxynucleotide is incorporated instead of a deoxynucleotide. This is a "chain termination" event, because there is a 3' H instead of a 3' OH group. The resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. Chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5' end with a fluorescent dye

Automated DNA sequencing (Dye termination sequencing):Most DNA sequencing is now automated. The Sanger method chain termination reactions are still used, but pouring, running, & reading polyacrylamide gels has been replaced by automated methods. Instead of labeling the products of all 4 sequencing reactions the same (with a radioactive deoxynucleotide), each dideoxynucleotide is labeled with a different fluorescent marker. When excitedwith a laser, the 4 different kinds of products are detected and the fluorescence intensity translated into a data “peak”. 

2. Chemical degradation method( Maxam and Gilbert): This method used chemical processes to terminate DNA strands. These fragment DNA pieces were then run through a gel to resolve the sequence order. The procedure for the whole process as follows:

• Denature a double-stranded DNA to single-stranded by increasing temperature.
• Radioactively label one 5' end of the DNA fragment to be sequenced by a kinase reaction using gamma-32P.
• Cleave DNA strand at specific positions using chemical reactions. For example, we can use one of two chemicals followed by piperdine. Dimethyl sulphate selectively attacks purine (A and G), while hydrazine selectively attacks pyrimidines (C and T). The chemical treatments outlined in Maxam-Gilbert's paper cleaved at G, A+G, C and C+T. A+G means that it cleaves at A, but occasionally at G as well.
• Now in four reaction tubes, we will have several differently sized DNA strands.
• Fragments are electrophoresed in high-resolution acrylamide gels for size separation.
• These gels are placed under X-ray film, which then yields a series of dark bands which show the location of radiolabeled DNA molecules. The fragments are ordered by size and so we can deduce the sequence of the DNA molecule.

Pros:Maxam-Gilbert sequencing was at one point more popular than the Sanger method. Purified DNA could be used directly, while the Sanger method required that each read start be cloned for production of single-stranded DNA.

Cons:It included difficulties scaling up, and the handling of X-rays and radiolabeling, which were harmful to technicians. 

Further development  is done to analysis and perform the genome level sequencing as this classical methods only hold few base pairs and it is difficult to analyses the whole genome or it will take too much time and proved expensive. 

Now we will discuss the Genome sequencing and advance method associated with it:

1. Genome sequencing: The technique in which the genetic information found in the DNA of anything from bacteria to plants to animals is decipher i.e, Sequencing involves determining the order of bases, the nucleotide subunits (adenine, guanine, cytosine and thymine, referred to by the letters A, G, C and T) found in DNA this technique is known as Genomic sequencing. In this technique genome has to be split into the fragments of a suitable size such that they can maintained within the vector.

 (Vector: It is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed). 

These genomic DNA fragments are cloned into a vector and each fragment is sequenced. For the assembly of the DNA fragments different approaches have been developed  these are known as Advance method of sequencing as described below: 

• Shotgun approach: In this method the DNA is first shredded into smaller fragments which can be sequenced individually. The sequences of these fragments are then reassembled into their original order, based on overlaps, ultimately yielding the complete sequence. In simple terminology DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads. 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. Haemophilus influenza is first sequenced by this technique.

Shotgun sequencing was one of the precursor technologies that was responsible for enabling full genome sequencing.

The two great outcomes of this technology was whole genome shotgun sequencing and Hierarchical Shotgun sequencing.

1. Whole genome shotgun sequencing: In the whole genome shotgun strategy , a high-molecular-weight DNA strand is sheared into random fragments, size-selected (usually 2, 10, 50, and 150 kb), and cloned into an appropriate vector. The clones are then sequenced from both ends using the chain termination method yielding two short sequences. Each sequence is called an end-read or read and two reads from the same clone are referred to as mate pairs. 

Paired-end tags (PET) (sometimes "Paired-End diTags", or simply "ditags") are the short sequences at the 5’ and 3’ ends of a DNA fragment which are unique enough that they (theoretically) exist together only once in a genome, therefore making the sequence of the DNA in between them available upon search (if full-genome sequence data is available) or upon further sequencing (since tag sites are unique enough to serve as primer annealing sites).

The original sequence is reconstructed from the reads using sequence assembly software. First, overlapping reads are collected into longer composite sequences known as contigs. Contigs can be linked together into scaffolds by following connections between mate pairs. The distance between contigs can be inferred from the mate pair positions if the average fragment length of the library is known and has a narrow window of deviation. Depending on the size of the gap between contigs, different techniques can be used to find the sequence in the gaps. If the gap is small (5-20kb) then the use of PCR to amplify the region is required, followed by sequencing. If the gap is large (>20kb) then the large fragment is cloned in special vectors such as BAC (Bacterial artificial chromosomes) followed by sequencing of the vector.

Contigs: A contig is a contiguous length of genomic sequence in which the order of bases is known to a high confidence level. 

A bacterial artificial chromosome (BAC): is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli.F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150-350 kbp 

Advantage:

The key advantage  of this approach argue that it is possible to sequence the whole genome at once using large arrays of sequencers, which makes the whole process much more efficient than other  traditional approaches.

 Disadvantage:

Although the technique quickly sequences large regions of DNA, its ability to correctly link these regions is suspect, particularly for genomes with repeating regions 

2. Hierarchical Shotgun sequencing:

The HS strategy involves an initial mapping step. A physical map is built using

clones with large inserts, such as BACs. The minimum tiling path of clones is

selected to cover the whole genome. Each clone is individually sequenced using

a shotgun strategy. A mixture of clone types is usually used in sequencing large

Genomes, also the different chromosomes may be separated and used to create

chromosome specific libraries.

 In this technique the amplified genome is first sheared into larger pieces (50-200kb) and cloned into a bacterial host using BAC. Because multiple genome copies have been sheared at random, the fragments contained in these clones have different ends, and with enough coverage finding a scaffold of BAC contigs that covers the entire genome is theoretically possible. This scaffold is called a tiling path.

(Scaffold: A scaffold is a portion of the genome sequence reconstructed from end-sequenced whole-genome shotgun clones. Scaffolds are composed of contigs and gaps).

In this process the overlapping clones can be identified by several way. The use of  Sequence-tagged site (STS) and by Chromosome walking.

A small radioactively or chemically labeled probe containing a sequence-tagged site (STS) can be hybridized onto a microarray upon which the clones are printed.In this way, all the clones that contain a partic