Essay !4 - About Sequencing Code : KSI0004
About Sequencinhg
Sangin Kim
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes falsely called primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.
1. DNA sequencing
DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. So far, most DNA sequencing has been performed using the chain termination method developed by Fredrick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as pyrosequencing are gaining an increasing share of the sequencing market. More genome data are now being produced by pyrosequencing than Sanger DNA sequencing. Pyrosequencing has enabled rapid genome sequencing. Bacterial genomes can be sequenced in a single run with several times coverage with this technique. This technique was also used to sequence the genome of James Watson recently
The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in fundamental research into why and how organisms live, as well as in applied subjects. Because of the key importance DNA has to living things, knowledge of DNA sequences are useful in practically any area of biological research. For example, in medicine it can be used to identify, diagnose, and potentially develop treatments for genetic diseases. Similarly, research into pathogens may lead to treatments for contagious diseases. Biotechnology is a burgeoning discipline, with the potential for many useful products and services.
1.1 sanger sequencing
In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymeraase an enzyme that replicates DNA. Included with the primer and DNA polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.
1.2 Pyrosequencing
Pyrosequencing which was developed by Pål Nyrén and Mostafa Ronaghi, has been commercialized by Biotage (for low-throughput sequencing) and 454 Life Sciences (for high-throughput sequencing). The latter platform sequences roughly 100 megabases [now up to 400 megabases] in a seven-hour run with a single machine. In the array-based method (commercialized by 454 Life Sciences), single-stranded DNA is annealed to beads and amplified via EmPCR. These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary base pairs. Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow.
1.3 Large scale sequencing
Whereas the methods above describe various sequencing methods, separate related terms are used when a large portion of a genome is sequenced. Several platforms were developed to perform exome sequencing (a subset of all DNA across all chromosomes that encode genes) or whole genome sequencing (sequencing of the all nuclear DNA of a human).
•Sequencing: determining the precise order of nucleotides in a DNA or RNA molecule
•Sanger dideoxy method
•Invented by Nobel Prize winner Fred Sanger
•Dideoxy analogs of dNTPs used in conjunction with dNTPs
•Analog prevents further extension of DNA chain
•Bases are labeled with radioactivity
•Gel electrophoresis is then performed on products
•Large-scale sequencing projects have led to automated DNA sequencing systems
•Based on Sanger method
•Radioactivity replaced by fluorescent dye
•Virtually all genomic sequencing projects use shotgun sequencing
•Entire genome is cloned, and resultant clones are sequenced
•Much of the sequencing is redundant
•Generally 7- to 10-fold coverage
•Computer algorithms are used to look for replicate sequences and assemble them
NGS(Next generation sequencing)
•Second-generation DNA sequencing
•Generates data 100x faster than Sanger method
•Massively parallel methods
•Large number of samples sequenced side by side
•Uses increased computer power and miniaturization
•454 Life Sciences pyrosequencing
•Illumina/Solexa sequencing
•SOLID/Applied Biosystems method
•454 sequencing system
•DNA is broken into small segments
•DNA is amplified using polymerase chain reaction (PCR)
•Light is released each time a base is added to DNA strand
•Instrument actually measures release of light
•Can handle only short stretches of DNA strand
•Third-generation DNA sequencing
•Sequencing of single molecules of DNA
•HeliScope Single Molecule Sequencer
•Single-stranded DNA fragments attached in array on glass slide
•Complementary strand synthesized
•Fluorescent tags monitored on microscope
•Computer assembles fragments into sequence
•Third-generation DNA sequencing
•Single Molecule Real Time sequencing
•Reactions carried out in nanocontainers (zero mode wave guides)
•Single-stranded DNA fragments attached
•Complementary strand synthesized
•Fluorescent tags monitored
•Computer assembles fragments into sequence
•Fourth-generation DNA sequencing
•Optical detection no longer used
•Ion torrent semiconductor sequencing
•Measures release of protons whenever a deoxyribonucleotide is added
•Silicon chip measures "pH"
•Extremely fast
•Less expensive
•Fourth-generation DNA sequencing
•Oxford Nanopore Technologies system
•Passes DNA through nanoscale biological pores
•Detector measures change in electric current
•Extremely fast
•Measures long chains of DNA
2. RNA sequencing
RNA is less stable in the cell, and also more prone to nuclease attack experimentally. As RNA is generated by transcription from DNA, the information is already present in the cell's DNA. However, it is sometimes desirable to sequence RNA molecules. While sequencing DNA gives a genetic profile of an organism, sequencing RNA reflects only the sequences that are actively expressed in the cells. To sequence RNA, the usual method is first to reverse transcribe the RNA extracted from the sample to generate cDNA fragments. This can then be sequenced as described above. The bulk of RNA expressed in cells are ribosomal RNAs or small RNAs, detrimental for cellular translation, but often not the focus of a study. This fraction can fortunately be removed in vitro, however, to enrich for the messenger RNA, also included, that usually is of interest. Derived from the exons these mRNAs are to be later translated to proteins that support particular cellular functions. The expression profile therefore indicates cellular activity, particularly desired in the studies of diseases, cellular behaviour, responses to reagents or stimuli. Eukaryotic RNA molecules are not necessarily co-linear with their DNA template, as introns are excised. This gives a certain complexity to map the read sequences back to the genome and thereby identify their origin. For more information on the capabilities of next-generation sequencing applied to whole transcriptomes see: RNA-Seq and MicroRNA Sequencing.
3. Protein sequcing
If the gene encoding the protein is known, it is currently much easier to sequence the DNA and infer the protein sequence. Determining part of a protein's amino-acidsequence (often one end) by one of the above methods may be sufficient to identify a clone carrying this gene.
4. Polysacharride sequencing
Though polysaccharides are also biopolymers, it is not so common to talk of 'sequencing' a polysaccharide, for several reasons. Although many polysaccharides are linear, many have branches. Many different units (individual monosaccharides) can be used, and bonded in different ways. However, the main theoretical reason is that whereas the other polymers listed here are primarily generated in a 'template-dependent' manner by one processive enzyme, each individual join in a polysaccharide may be formed by a different enzyme. In many cases the assembly is not uniquely specified; depending on which enzyme acts, one of several different units may be incorporated. This can lead to a family of similar molecules being formed. This is particularly true for plant polysaccharides. Methods for the structure determination of oligosaccharides and polysaccharidesinclude NMR spectroscopy and methylation analysis.
Reference
1. https://www.youtube.com/watch?v=jFCD8Q6qSTM
2. Brook biology of microorganisms 14th edition.
