DNA ( Deoxy ribo Nucleic Acid) contains all the information required for a cell to survive and this is present in the form of four bases, represented by the letters A, C, G and T. These letters represent four Nitrogenous bases present in the DNA,which are Adenine,Cytosine,Guanine and Thymine. The order of bases on a DNA strand is the DNA sequence. The sequence of DNA constitutes the heritable genetic information in nuclei, plasmids, mitochondria, and chloroplasts that forms the basis for the developmental programs of all living organisms. Determining the sequence of DNA is vital for understanding any kind of research (applied and basic or fundamental) and latest techniques developed for sequencing revolutionized the field of biomedical sciences in finding suitable treatment for diseases. DNA sequencing is all about determining the exact order of the bases A, T, C and G within a piece of DNA.
DNA sequencing was performed by many ways in the olden days ,but the most famous one being the dideoxy or chain termination method – developed by Fred Sanger in 1977 ,for which he was awarded his second Nobel prize.As the time passed by and technology improved ,leading to more sophisticated sequencers.Thanks to these latest developments ,today we have complete genome sequences ( you will get the complete list of sequenced Genomes on Genome News Network ) of various metazoans including humans.
Automated DNA sequencer:
A DNA sequencer can be defined as an instrument used to automate the DNA sequencing process. Modern automated DNA sequencing instruments (called DNA sequencers) are capable of sequencing multiple samples in a batch (run) and can perform many runs a day. These sequencers perform only the size separation and peak reading; the actual sequencing reaction(s)must be performed separately.
how DNA Sequencing works ???
DNA sequencing reactions are just like the PCR reactions The reaction mixture consists of the template DNA, free nucleotides, an enzyme (Taq polymerase) and a ‘primer’. A Primer is a small piece of single-stranded DNA about ~20 nucleotide long that can hybridize to one strand of the template DNA.The reaction is started by heating to separate the template DNA strands and later at an appropriate temperature primer binds to one strand helping taq polymerase to elongate the primer.This results in completion of a new strand in a manner that nucleotides added to the growing DNA strand are complementary to those in the template strand (A pairs with T and G with C ).
These steps are exactly like for PCR reactions except for the use of dideoxyribonucleotide. This is just like regular DNA, except it has no 3′ hydroxyl group – once it’s added to the end of a DNA strand, there’s no way to continue elongating it.But one important thing to note is that most of the nucleotides used are regulars ones ,only few of them will be dideoxy nucleotides. Sequencing is achieved by including in each reaction a nucleotide analogue that cannot be extended and thus acts as a chain terminator.
Four reactions are set up, each containing the same template and primer but a chain terminator specific for A, C, G or T. Because only a small amount of the chain terminator is included, incorporation into the new DNA strand is a random event. Each reaction therefore generates a collection of fragments, but every DNA strand will end at the same type of base (A, C, G or T). Important thing to note here is that each of the four reactions contain different fluorescent labels allowing DNA strands terminating at each of the four bases to be identified after being separated by gel electrophoresis (four reactions are mixed and loaded). The machine enables the separation of DNA molecules according to size even if they differ in length by only a single nucleotide(‘capillary electrophoresis’). As the DNA strands pass a specific point, the fluorescent signal is detected by the UV laser in the machine and hence the base identified.The reading for sequence is also completely automated.
Current methods can directly sequence only relatively short (~1000 nucleotides long) DNA fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide. So in order to sequence long fragments of DNA ( as in the case of sequencing of whole animal genomes) a process called as shotgun sequencing is widely employed.
Shotgun involes cutting (with restriction enzymes) large DNA fragments into shorter DNA fragments. and later the fragmented DNA is cloned into a DNA vector, usually a bacterial artificial chromosome (BAC), and amplified in Escherichia coli.
The amplified DNA can then be purified from the bacterial cells .These short DNA fragments purified from individual bacterial colonies are then individually and completely sequenced and assembled electronically into one long, contiguous sequence by identifying 100%-identical overlapping sequences.
Further reading :
For a detailed view of how sequencing is performed see this page from University of Michigan