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The techniques used to study molecular biology of nucleic acids are:- 1. Gel electrophoresis 2. Use of restriction endonucleases 3. DNA hybridization 4. Hybridization probes 5. Isolation of specific segments of DNA 6. DNA cloning 7. Cloning DNA in plasmid vectors 8. Vector DNA can be introduced into host organisms by transformation 9. Libraries of DNA molecules can be created by cloning and a few others.
Technique # 1. Gel electrophoresis:
In which electrophoresis through a gel separates DNA and RNA molecules according to size.
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Technique # 2. Use of restriction endonucleases:
Which cleaves i.e., splits or cuts DNA molecules at particular sites.
Technique # 3. DNA hybridization:
Which can be used to identify specific DNA molecules.
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Technique # 4. Hybridization probes:
Which can identify electrophoretically separated DNAs and RNAs.
Technique # 5. Isolation of specific segments of DNA:
Which is used for separation of specific segments of DNA from much larger DNA molecules followed by their selective amplification? Hence the DNA can be sequenced.
Technique # 6. DNA cloning:
This is the ability to construct recombinant DNA molecules and maintain them in cells. This process, however, involves a vector.
Technique # 7. Cloning DNA in plasmid vectors:
In it for example plasmid vector has a unique recognition site for EcoRI. Treatment with the appropriate restriction enzyme would line size the plasmid. A target DNA is cleaved with a restriction enzyme to generate potential insert DNAs. Vector DNA which has been cut with the enzyme is mixed with these insert DNAs and DNA ligase is used to link or join the compatible ends of two DNAs.
Technique # 8. Vector DNA can be introduced into host organisms by transformation:
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Transformation is a process in which host organism can take up DNA from its environment. However, transformation is a relatively inefficient process.
Technique # 9. Libraries of DNA molecules can be created by cloning:
A DNA library is a population of identical vectors that each contains a different DNA insert. Different types of libraries are made using insert DNA from different sources.
To enrich for coding sequences in the library a cDNA library is used. Instead of starting with genomic DNA, mRNA is converted into DNA sequence the process which permits it is called reverse transcription, and is done with a special DNA polymerase (reverse transcriptase) which can make DNA from RNA template.
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When treated with reverse transcriptase mRNA sequences can be converted into double stranded DNA copies which are known as cDNAs (for copy DNAs or complimentary DNA). These fragments can be ligated into the vector.
Technique # 10. Hybridization can be used to identify a specific clone in a DNA library:
The process by which a labelled DNA probe is used to screen a library is known as colony hybridization.
Technique # 11. Use of Chemically synthesized oligonucleotides:
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An oligonucleote is a short, synthetic, single-stranded DNA molecule one of a type which is used as a primer in DNA sequencing or PCR.
Technique # 12. Polymerase Chain Reaction with a PCR machine or Thermocycler:
Which amplifies DNAs by repeated rounds of DNA replication in vitro? PCR employs the enzyme DNA polymerase which directs the synthesis of DNA from deoxynucleotide substrates on a single stranded DNA template.
DNA polymerase synthesizes DNA in a 5 to 3 end direction and can add nucleotides to the 3′ end of a custom designed oligonucleotide. DNA polymerase can use the oligonucleotide as a primer and elongate it in a 5 to 3 direction to generate an extended region of double stranded DNA.
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It should be noted that to begin within the first step of the PCR the DNA template is denatured by heating and annealed or attached with synthetic oligonucleotide primers. DNA polymerase is added. It is accompanied heat and repeat. Finally DNA will increase in abundance with each subsequent cycle of the chain reaction.
Technique # 13. Nested sets of DNA fragments of several nucleotide sequences:
Nested sets of DNA molecules are created by two ways. In first procedure DNA molecules are radioactively labelled at their 5′ terminal and are then subjected to break preferentially at Gs, Cs, Ts, or As. However, this chemical procedure is no longer in wide use. The second procedure chain-terminating nucleotides are used. The modern automatic sequencing machines called sequenators are based on this technology.
Technique # 14. Shotgun sequencing a bacterial genome:
The bacterial species Hemophilus influenza was the first organism to have a complete genome sequence and assembly. The genome of this bacterium is composed of 1.8 megabase pairs (mb) of DNA. The H influenza genome was randomly sheared into many random fragments with an average size of 1 kb.
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These pieces of genomic DNA were cloned into a plasmid recombinant DNA vector. DNA was prepared from individual recombinant DNA colonies and sequenced separately on Sequenators employing dideoxy method. This method is known as shotgun sequencing.
Technique # 15. Use of shotgun strategy for partial assembly of large genome sequences:
Sophisticated computer programmes have been prepared which assemble the short sequences from random shotgun DNAs into larger contiguous sequences known as contigs. Short contigs are assembled into larger scaffolds which are typically 1 – 2 Mb long.
Technique # 16. Use of paired-end strategy for the assembly of large genome scaffolds:
Special cloning vector known as (BAC) or bacterial artificial chromosome. The use of BACs is used for the assignment of multiple contigs into a single scaffold of several megabases.
Technique # 17. Genome – wide analysis:
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A variety of bioinformatics tools are required to identify genes and know the genetic composition of complex genomes. Now a day’s computer programmes have been developed which identify protein coding genes through a variety of sequence criteria including the occurrence of extended open reading frames or ORF (a series of codons that is or could be a gene), flanked by appropriate 5′ and 3′ splice sites. A limitation of current gene finder programme is the failure to identify promoters.
Therefore, most important method for validating predicted protein coding gives and identifying those missed by current gene finder programme is the use of cDNA sequence data cDNAs are produced by reverse transcription from mature mRNAs and thus represent bonafide or real exon sequences. Thus cDNA is used to generate EST or expressed sequence tag, data, which is a short sequence read from a larger cDNA.
Technique # 18. Comparative genome analysis:
A noted finding of comparative genome analysis is the high degree of syntery i.e. conservation of genetic linkage between distantly related animals. The most common used genome tool is BLAST or basic local alignment search tool. Searching a genome or many genomes, for all the predicted protein sequences which are related to be called query sequence.
