Gene library can be created by extracting the DNA and by using endonucleases to cut the DNA of specific organism. Next step is to insert the cutting (purified) fragments into the vector. Then the ligation of purified DNA fragments into the vector according to size of genomics library requirement. These clones can be detected with the help of the DNA prob. To obtain a desired gene from recombinants following formula can be used.
N= no of recombinants
P=required gene probability in library
f= genome fraction in one insert
The cDNA library is created by using the mRNA. The mRNA is taken from eukaryotic cells and cDNA is created by using the reverse transcriptase. The eukaryotic mRNA has polyA tails and introns but the cDNA has no polyA tail and introns so these cDNA can be introduced in prokaryotic cells. The first step of cDNA library construct is the extraction of mRNA. There are many methods to extract the mRNA like trizol extraction and column purification. The oligomeric -dt nucleotide coated resins are used in this method to which only mRNA having the polyA tail can bind. All other RNAs are eluted out by eluting buffer. The warming of media can separated the mRNA from oligomeric -dts. Once the mRNA is purified the reverse trancriptase is used to make complementary DNA strand of mRNA. The double stranded DNA is synthesized from that single stranded cDNA by polymerase. The DNA is being cut by endonucleases and cloned into the bacterial plasmid.
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Subcloning is a procedure which is being used to move a target gene of interest from a parent vector to another vector to study its detail functions. The target gene is excised by using the restriction enzymes from the parent. The gene is amplified then by using PCR. The restriction enzymes create sticky ends which help in ligation of target genes into the plasmid. The self ligation of the destination vector is prevented by using the alkaline phosphate. Then the target gene should mix in the vector in the presence of ligand. Mostly E. coli is used for transformation. Then E. coli is being grown and after replication each bacterial cell has many copies of the plasmid and DNA is harvested.
The markers are used to select only the transformed colonies. Mostly the antibiotics are used as marker.
Plasmid (PUC19, ColE1), Phage (Lambda, M13 and P1), Phagemid (pBluescript series), Cosmid, Fosmid, BACs (Bacteria artificial chromosome, YACs ( yeast artificial chromosome are most commonly used as cloning vector for genomics.
The size of Plasmid vector is 20kb, cosmid 45kb , λ-phage10-15kb, YAC 1000 kb, BAC can take up to 300 kg but its size is 100kb, Lambda phage is 45 kbp linear chromosome and can hold upto 9-23 kbp.
The Genomic libraries can be used to find out the whole genomic sequence of given organism. Also these libraries provide genomic sequence for creation of transgenic animals by genetic engineers.
The cDNA libraries can be used to trace out eukaryotic genomes and also to expose eukaryotic genes in prokaryotes cDNA.
Colony picker are being used for colony transfer from an agar plate. This process is very entrust process so tremendous care be bothered for maximum accuracy. Now many advanced colony picker are accessible for DNA sequencing, library screening, colony management, protein evolution and protein engineering.
Northern blotting is the technique used for the study if gene expression.
Northern blotting has these steps.
Extraction and purified RNA from through oligo (dt) from a homogenized tissue.
Isolation of mRNA contain only the polyA tail through oligo (dt) cellulose chromatography
Separation of RNA by gel electrophoresis.
Transfer of RNA to nylon membrane via vacuum or capillary blotting system. The nylon membrane has positive charge which has affinity for negative charged nucleic acid.
The transfer buffer contains formamide used for transfer because it decreases the probe-RNA's annealing temperature and prevent it from degradation by high temperatures.
After that a probe is labeled to hybridize the RNA on the membrane.
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Next step is membrane washing to make it sure that the probe is bounded specifically.
The hybrid signals can be detected by X-ray film and measured by densitometry
The gene that is not of interest can be detected after determination by microarrays.
The Sanger's method is also known as the chain termination or dideoxy sequencing. The basic principle of Sanger's is to add dideoxynucleotides besides the normal nucleotides present in DNA. The dideoxynucleitides are the same except that these nucleotides have hydrogen group on it 3' end in place of hydroxyl group. When these nucleotides are being added to any DNA sequence then it will block the further nucleotide addition. This is because of phosphodiester bond cannot established between the coming nucleotide and dideoxynucleotide so there would not be further nucleotide addition. For sequence detection the double stranded DNA is converted into the single stranded DNA. The primer is designed to amplified the one of ssDNA this primer are radioactively fluorescent and should have the ability if chain termination. Then the sample is divided into different four sequencing reactions which have all four nucleotides (A, T, C and G). Thus in each reaction one nucleotide is being added which are terminating nucleotides so the final product after extension have different chain length. The new strands then are denatured by heat and are separated according to their size in gel electrophoresis. These strands would be on four different lines and then they can be visualized and can be read on the X-ray film
Pyrosequancing is a DNA sequencing technique. In this sequencing method the complementary strand is synthesized against the single stranded DNA. This sequencing method actually based on the enzymatic DNA polymerase activity and compared to other chemiluminescent enzyme. In this sequencing the polymerase adds the nucleotide in each step and the added nucleotide is being identified. The light is used as the detector because in every step when the complementary nucleotide is added there is light emission that provides the clue that which nucleotide is being added.
The formation of double helix between the sense and anti sense RNA is common naturally and forms the dsRNA. These dsRNA have the ability of suppression of particular gene expression. This suppression ability of dsRNA of particular gene which is complementary to its own sequence is known as the RNA interference. Normally there are many dsRNA are present in cytoplasm and these are cut by the enzyme called as dicer into small dsRNA. When there is complementary sequence of mRNA with these dsRNA, then this mRNA is ruined.