Thursday, June 20, 2013

Hybridization uses to identify a clone with a particular DNA Segment

Use of hybridization to identify a clone with a particular DNA segment:

Library and Screening of Library.

A collection of different recombinant clones is called library.

Genomic library:

Due to availability of restriction enzymes and cloning vectors the entire genome of organism can be individually packed into a vector. A genomic library is a collection of fragments of total ds-DNA (as obtained by cutting with restriction endonuclease) of a cell line or tissue.

These fragments are cloned and amplified in host; these fragments represent the whole genome. If the digestion is allowed to go to completion, the gene of interest is fragmented— and is not available in intact form. To avoid this, a partial digestion is performed in which either the amount or the time of action of the enzyme is limited. This will only cleave a fraction of the restriction sites on any one DNA molecule, thus producing fragments of about 20,000 base pairs. Enzymes that cut very frequently (that is, enzymes that recognize four-base-pair sequences) are generally used for this purpose to randomly collect fragments. This ensures that the gene of interest is contained, intact, in some fragment. BAC, YAC and P1 vectors are generally used for making genomic library which can accept large fragment of DNA, so there is chance of getting intact eukaryotic mRNA-encoding gene.

Note: Klenow polymerase is a normal DNA polymerase whose 5’ to 3’ exonuclease activity is destroyed by treating it with subtilisin.

Collection of bacteria or hosts harboring a particular segment of genome forms the clone. And the entire clone that contribute the whole genomic segments forms genomic library.

A cDNA library: 

It consist cDNA copies of the population of mRNAs in tissue. For example, reticulocyte mRNA is composed of genes encoding α-globin and β-globin chains of hemoglobin. This mRNA can be used as a template to make a complementary dsDNA (cDNA) molecule using the enzyme reverse transcriptase. The resulting ds cDNA fragments are then inserted into suitable vector and cloned, creating a population of clones called cDNA library. (mRNA is isolated from tRNA and rRNA by the presence of its poly (A) tail. cDNA can be amplified by cloning or by the polymerase chain reaction. It can be used as a probe to locate the gene that coded for the original mRNA (or fragments of the gene) in mixtures containing many unrelated DNA fragments. Because cDNA has no intervening sequences, it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria. These special plasmids contain a bacterial promoter for transcription of the cDNA, and a Shine-Dalgarno sequence that allows the bacterial ribosome to initiate translation of the resulting mRNA molecule.

Fig: Synthesis of double stranded cDNA from mRNA template.
Screening of Library:

Various probes are used for searching specific genes in the library. These probes are small piece of DNA or RNA labeled with a 32P-containing nucleotide or fluorescently labelled nucleotide. This probe should recognize and hybridize with a complementary sequence of the target DNA. Probes are used to identify which clone of a library or which band on a gel contains the target DNA.

Due to danger of radioactive substance nowadays biotinylated nucleotide are used. The avidin is attached to fluorescent dye. Thus, a DNA fragment (displayed, for example, by gel electrophoresis) that hybridizes with the biotinylated probe can be made visible by immersing the gel in a solution of dye- coupled avidin. After washing away the excess avidin, the DNA fragment that binds the probe is fluorescent.

If the sequence of all or part of the target DNA is known, single-stranded oligonucleotide probes of 20–30 nucleotides can be synthesized that are complementary to a small region of the gene of interest. If the sequence of the gene is unknown, the amino acid sequence of the protein—that is, the gene product—may be used to construct a probe. Short, ssDNA sequences (15–30 nucleotides) are synthesized, using the genetic code as a guide. Because of the degeneracy of the genetic code, it is necessary to synthesize several oligonucleotides. [Note: Oligonucleotides can be used to detect single-base changes in the sequence to which they are complementary. In contrast, cDNA probes contain many thousands of bases, and their binding to a target DNA with a single-base change is unaffected.

RNase H produces nick in the RNA strand exposing its 3’-OH which are used by DNA polymerase as a primer to replace the RNA with a second strand of cDNA.

The probe is designed to be complementary to the region of gene with minimal degeneracy, i.e region with fewest possible codons for amino acids, tow codons at most. Because this site will provide maximum chance of complementary base pairing.

ssDNA, produced by alkaline denaturation of dsDNA, is first bound to a solid support, such as a nitrocellulose membrane. The immobilized DNA strands are prevented from self-annealing, but are available for hybridization to an exogenous, single-stranded, radiolabeled DNA probe. The extent of hybridization is measured by the retention of radioactivity on the membrane. Excess probe molecules that do not hybridize are removed by washing the filter and, therefore, do not interfere. This is the basis of blotting technique.

cDNA probes are used to detect DNA fragments on Southern blot transfers and to detect and quantitate RNA on Northern blot transfers. Sometimes no amino acid sequence information is available to guide the synthesis of a probe for direct detection of the DNA of interest. In this case, a gene can be identified indirectly by cloning cDNA in an expression vector that allows the cloned cDNA to be transcribed and translated. A labeled antibody is used to identify which bacterial colony produces the protein and, therefore, contains the cDNA of interest. [Note: A library created using expression vectors is called an expression library.]
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