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INTRODUCTION
What is PCR ? PCR is a laboratory procedure in which millions copies of specific DNA are made. This method is developed by Kary Mullins in 1980s What is it all about ? its a chain reaction involving DNA polymerase and short piece of single stranded DNA (called primer) to synthesize a new DNA strand complementary to the template strand (called amplicon) Note that PCR cannot work without a primer because it needs a template strand (rich in 3 OH group) to which it can add nucleotide and create a self sustaining DNA strand
Kary Mullis
Once synthesize has been completed,the whole mixture is heated again to 95C to melt the newly formed DNA complexes. This results in twice the amount of template available for the next round of replication. Repeated heating and cooling quickly amplifies the DNA segment of interest. Roughly, 1 million of copies are made after 20 cycles.
GENE BANK
Definition :
A collection of seeds & other plants reproductive material, primarily of cultivated plants & their wild relatives.
Represent as far as possible the gene pools of crop plants, that is, the genetic basic of agriculture and horticulture.
Mandate :
To secure the conservation of these collected plants genetic resources & provide access to them.
Gene bank have primarily been concerned with : (a) Ex-situ Off-site conservation Vegetatively propagated plants growing in donal archives at different sites Involed dried & frozen seed samples Process involved are : (i) in-vitro preservation (ii) cryopreservation i. In-vitro preservation - of tiny growing tips in test tube - example : potatoes, green peas, etc
ii .
(b) In-situ On farm, in nature Preservation of some species, plant associations & forms of cultivation Example : meadows, or wild crop relatives in marginal natural biotopes.
(b) Tissue bank Conserved through particular light & temperature arrangements in nutrient medium. Used to preserve seedless plants & plants which reproduced sexually. (c) Cryobank A seed or embryo is preserved at very low temperature. Usually preserved in liquid nitrogen at -196c Helpful for the conservation of species facing extinctions. (d) Pollen bank Stored of pollen grains. Make plants which are facing extinction in the present world with one set of chromosomes.
(e)
Field gene bank Method of planting plants for the conservations of gene. Construct ecosystem artificially. Can compare the differences among plants of different species & can study it in details. Need more lands, adequate soil, weather, etc. Example : germ plasma of important crops.
GENE LIBRARY
A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using the enzyme, DNA ligase. the vector DNA can be taken up by a host organism- commonly a population of Escherichia coli or yeast, with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.
There are several kinds of vectors available with various insert capacities. Generally, libraries made from organisms with larger genomes require vectors featuring larger inserts, thereby fewer vector molecules are needed to make the library. Researchers can choose a vector also considering the ideal insert size to find a desired number of clones necessary for full genome coverage. Genomic libraries are commonly used for sequencing applications. They have played an important role in the whole genome sequencing of several organisms, including the human genome and several model organisms. Genomic libraries are also utilized in comparison studies between differing species.
The first DNA-based genome ever fully sequenced was achieved by two-time Nobel Prize winner, Frederick Sanger, in 1977. Sanger and his team of scientists created a library of the bacteriophage, phi X 174, for use in DNA sequencing. The importance of this success contributed to the ever-increasing demand for sequencing genomes to research gene therapy. Teams are now able to catalog polymorphisms in genomes and investigate those candidate genes contributing to maladies such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, rheumatoid arthritis, and Type 1 diabetes. These are due to the advance of genome-wide association studies from the ability to create and sequence genomic libraries. Prior, linkage and candidate-gene studies were some of the only approaches.
Construction of a genomic library involves creating many recombinant DNA molecules. An organism's genomic DNA is extracted and then digested with a restriction enzyme. For organisms with very small genomes (~10 kb), the digested fragments can be separated by gel electrophoresis. The separated fragments can then be excised and cloned into the vector separately. However, when a large genome is digested with a restriction enzyme, there are far too many fragments to excise individually. The entire set of fragments must be cloned together with the vector, and separation of clones can occur after. In either case, the fragments are ligated into a vector that has been digested with the same restriction enzyme. The vector containing the inserted fragments of genomic DNA can then be introduced into a host organism.
Below are the steps for creating a genomic library from a large genome.
1. 2. 3.
Extract and purify DNA. Digest the DNA with a restriction enzyme. This creates fragments that are similar in size, each containing one or more genes. Insert the fragments of DNA into vectors that were cut with the same restriction enzyme. Use the enzyme DNA ligase to seal the DNA fragments into the vector. This creates a large pool of recombinant molecules. These recombinant molecules are taken up by a host bacteria by transformation, creating a DNA library.
4.