THE POLYMERASE CHAIN REACTION & GENE BANK &

GENE LIBRARYBASIC GENETIC

FGS0054

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 1980’s What is it all about ? – it’s 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

COMPONENTS OF POLYMERASE CHAIN REACTION

A template DNA strand – to create a complementary strand DNA polymerase – to amplify and synthesize the individual DNA

strands of the template provided A primer – provides 3’-OH group to polymerase and adds nucleotides

to create individual DNA strand Nucleotides – Contains single unit of the bases A,G,T and C

HOW POLYMERASE CHAIN REACTION WORKS ?

PCR basically works in four major steps :

Denaturation – it is carried out at 95ºC. DNA exist in nature as double stranded molecule linked together by hydrogen bonds. To be able to copy it,the DNA need to be separated into single strands

Annealing – it is carried out around 55ºC-60ºC. Although the tempertaure is lowered,an excess amount of primers prevents the denatured DNA from reforming the double helix. The primers attach or ‘anneal’ to their matching sequence on the original DNA strand

Extension – it is carried out around 72ºC. Taq DNA polymerase binds to the annealed primer. Taq polymerase works its way along the DNA,adding complementary nucleotides using the dNTPs and other components in the reaction mix. This completes the replication process

Once synthesize has been completed,the whole mixture is heated again to 95ºC 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.

APPLICATIONS OF POLYMERASE CHAIN REACTION

Infectious disease – in HIV cases, PCR can detect as little as one viral genome among the DNA of over 50000 host cells. Infections,can be detected earlier,donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified

Research – used in the more traditional process of cloning. It can extract segments for insertion into a vector from a larger genome,which may be only available in small quantities. Using a single set of ‘vector primers’,it can extract fragments that have been already inserted into vectors.

BENEFITS OF POLYMERASE CHAIN REACTION

Amplifies DNA – the main advantage of PCR in the field of forensic science is that scientists can utilize it for amplifying it or making several copies of parts of the DNA that widely vary between different people,known as VNTRs. This device can easily amplify whichever small amounts of the DNA attained. Through comparing the various VTNRs,scientists can determine if the DNA sample is a match as compared to the defendant DNA

Better HIV test – PCR also quite useful in HIV testing as it is more sensitive and accurate. Instead of searching for the presence of HIV antibodies, PCR directly looks for pro-viral DNA. The complexity of this test makes it 98% accurate at 29 days from an assumed exposure or contact.

LIMITATION OF POLYMERASE CHAIN REACTION

Primers need to be used with care. If they don’t exactly match the template, there are possibilities of strand mutation. Moreover,specific attention need to be given to the annealing step since the primer tends to (sometimes) anneal to themeselves leading to defective DNA synthesis

The most crucial challenge while attempting a PCR is hygiene. Toxins, blood drops and other substances may contaminate the DNA strand and gives you misleading results, especially when used in forensic laboratory

The presence of divalent cations and nucleotides must exactly match the synthesis specification, or a non-specific DNA amplication will take place

GENE BANK

Definition : A collection of seeds & other plants reproductive material,

primarily ofcultivated 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 . Cryopreservation - at extreme low pressure - example : growing tips of hops, etc

(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.

Responsibilites of Gene Bank

• Acquiring the most important materials for their region without undue duplication.

• Successfully treating and storing samples.• Maintaining & regenerating the collection without losing or

changing genetic materials.• Providing the security through backup collections.• Responsible for registring, studying, describing &

documenting its collection ( making both information & plant materials available to researchers & other interested users ).

Types of Gene Bank

(a) Seed bank• Preserved dried seeds by storing them at a very low

temperature.• Example : spores & pteridophytes.• Seedless plants ( eg: tubercrops ) preserved by citation.• Largest seed bank in world; Millennium Seed Bank House

at the Welcome Trust Millennium Building ( WTMB ), West Sussex.

(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 -196ºc• 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.

Conserving & Sharing Genetic Diversity

• Conserve living samples of the world’s huge diversity of crop varieties & their wild relatives.

• Ensure that genetic resources that underpin food supply are both secure in the long term & available for the use by formers, plant breader’s & researcher.

• Maintain clonal & seed collections & houses are of the world’s leading herbarium collections.

• CIP’s germplasm is available for free to developing countries.

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.

WHAT IS GENE LIBRARY

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.

HISTORY OF GENE LIBRARY

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.

HOW GENE LIBRARY ARE CONSTRUCTED

1. Extract and purify DNA.2. Digest the DNA with a restriction enzyme. This creates

fragments that are similar in size, each containing one or more genes.

3. 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.

4. These recombinant molecules are taken up by a host bacteria by transformation, creating a DNA library.

Below are the steps for creating a genomic library from a large genome.

Below is a diagram of the above outlined steps