Identification of genes : Identification of genes Presented by-
Manipal college of pharmaceutical sciences Contents : Contents Techniques used in gene identification
Separation of DNA fragments using AGAROSE/
POLYACRYLAMIDE Gel Electrophoresis-
Methods used in gene identification
1. Screening of genomic & cDNA library-
A. Screening by DNA hybridization
B. Screening by plaque / colony hybridization
C. Screening by immunological assay-
D. Functional / genetic complementation-
2. Transposon & Insertional mutagenesis 1. Separation of DNA fragments using AGAROSE/ POLYACRYLAMIDE Gel Electrophoresis- : 1. Separation of DNA fragments using AGAROSE/ POLYACRYLAMIDE Gel Electrophoresis- When genomic DNA is digested with a restriction enzyme, it is cleaved into different segments.
The segments of different sizes can be separated through gel electrophoresis.
Gel electrophoresis involves the movements of DNA fragments or molecules under a high voltage electric current. In gel, the negatively charged DNA fragments move towards the positive electrode at a rate inversely proportional to their length. Slide 4: After the electric field is applied for a certain period, DNA fragments with different length will separated. Heavier fragments will remain closer to the loading site & lighter fragment will move away.
Migration rate of DNA is inversely proportional to the log molecular weight.
Fragments separated by the size of the gene segment.
Smaller fragments move faster than larger.
Buffer solution provides a means of transmission for electrical current but also keeps DNA samples in place in wells in the gels. 2. Southern blotting- : 2. Southern blotting- The name of this technique is derived from the name of its inventor E.M. Southern & the DNA-RNA hybridization that forms its basis.
In this technique DNA sample is digested with a restriction enzyme & the digested sample is gel electroforsed.
The DNA bands in the gel are denatured into single strands with the help of an alkali solution.
The gel is then placed on the top of a buffer saturated filter paper, which is placed on a solid support, with its 2 edges immersed in the buffer solution.
A sheet of nitrocellulose paper is placed on top of the gel & a stock of paper on top of this membrane.
The buffer moves due to capillary action from the bottom filter paper through the gel to the nitrocellulose membrane carrying with it the denatured DNA present in the gel. Slide 7: Thus DNA becomes trapped in the nitrocellulose membrane. This process is known as blotting & requires several hours to complete.
Nitrocellulose membrane is then kept at 800 C for 2-3 hrs.to fix the DNA permanently on the membrane.
Then the membrane is treated with a solution containing 0.2% each of ficoll (an artificial polymer of sucrose), PVP, & bovine serum albumin along with an irrelevant nucleic acid like t-RNA. This treatment prevents non specific binding of radioactive probe.
The pretreated membrane is placed in a solution of radioactive probe, which will hybridize to the complementary ssDNA on the membrane. This is known as hybridization reaction.
The membrane may then be washed to remove any unbound DNA.
Finally X-ray film is exposed to the hybridized membrane to get an autobiography. Methods for gene identification- : Methods for gene identification- 1. Screening of genomic & cDNA library-
DNA library is a collection of cloned DNA fragments.
The genomic library contains DNA fragments representing the entire genome of an organism.
The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell.
The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library is called as screening Slide 11: Screening can be done by the following methods.
A) Screening by DNA hybridization
B) Screening by colony & plaque hybridization
C) Screening by immunological assay
D) Screening by functional /genetic complementation A. Screening by DNA hybridization : A. Screening by DNA hybridization The presence of a target nucleotide sequence in a DNA sample can be determined with a DNA probe.
This procedure is called DNA hybridization
The steps involved are same as in southern blotting
B. SCREENING BY PLAQUE / COLONY HYBRIDIZATION
This method also utilizes a DNA probe. This is used for colony hybridization.
The procedure involved is as follows
1. Transfer some of DNA in plaque / colony to a nylon / nitrocellulose membrane. Because plaques are areas of lysed bacteria, the phase DNA is directly available & will bind to the membrane when top of the petridish.
This can be achieved by soaking in sodium dodecyl sulphate & protease. Slide 13: 2. The DNA on the membrane is denatured with alkali to produce single strands; deprotenised & is bonded to the membrane by baking or UV radiation.
3. The membrane is then immersed in a solution containing a nucleic acid probe, which is usually radioactive & incubated to allow probe to hybridize to its complimentary sequence.
4. After hybridization, the membrane is washed extensively to remove unhybridised probe.
5. The region where the probe has hybridized is visualized by exposure to X-ray film.
6. A colony on the master plate that corresponds to the region of a positive response on the X- ray film is identified. Cell from the positive colony on the master plate are sub cloned because the carry the desired DNA.
Thus the identification of gene of interest from the whole genomic library is achieved. C. Screening by immunological assay- : C. Screening by immunological assay- This method is used when a DNA probe is not available.
In this method all the clones of the library are grown separately on a plates. A sample of each colony is transferred to a known position on a matrix, where the cells are lysed & the released proteins are attached to the matrix.
The matrix with the bound proteins is treated with an antibody (primary antibody) that specifically bounds to the protein encoded by the target gene.
Following the interaction of primary antibody with the target protein (antigen), any unbound antibody is washed away, and matrix is treated with a second antibody (secondary antibody) that is specific for primary antibody.
In many assay systems, the secondary antibody has an enzyme, such as alkaline phosphatase, attached to it. After the matrix is washed, a colorless substrate is added.
If the secondary antibody has bound to the primary one, the colorless substrate is hydrolyzed by the attached enzyme & produces a colored compound that accumulates at the site of action. D. Functional / genetic complementation- : D. Functional / genetic complementation- This is another way of identifying genes that encode enzymes.
The procedure involved transforming host cell that have a particular genetic defect with plasmids of a DNA library constructed from a normal organism & selecting the transformed cell that function normally 2. Transposon & Insertional mutagenesis- : 2. Transposon & Insertional mutagenesis- Identification of a gene whose phenotypic effect is known, but the gene product is unknown, can be done by using transposable elements (transposon tagging).
Such genes include those for morphological traits like dormancy, disease resistance, and photoperiodicity.
In this transposon works as a gene tag. following steps are involved in this procedure
A) Clone a known gene with a phenotypic effect
B) TE is transposed to this gene to get an unstable allele
C) This unstable allele is cloned & TE is isolated from this unstable allele ( This is to select a TE which can produce unstable allele.
D) This TE is transposed to a gene of interest with known phenotypic effect , to produce unstable allele.
E) The DNA is extracted from this mutant.
F) TE sequence is used as a probe to identify the mutant gene carrying inserted TE. Thus we can identify the gene of interest. References: : References: Walker J.M., Gingold E.B, “Molecular Biology & Biotechnology”, 2nd edi, 1993, panima publishing educational book agency, New Delhi, 144.
MOLBIO: fundamentals of molecular biology”, 1st edi. 2005, Himalaya publishing house, Meerut, page no.-20-28.
http://www.molecular-plant-biotechnology.info/molecular-probes-and-gene-libraries/construction-and-screening-of-genomic-and-cDNA-libraries.htm Slide 20: Thank you…