Algae, Tree, Herbs, Bush, Shrub, Grasses, Vines, Fern, Moss, Spermatophyta, Bryophyta, Fern Ally, Flower, Photosynthesis, Eukaryote, Prokaryote, carbohydrate, vitamins, amino acids, botany, lipids, proteins, cell, cell wall, biotechnology, metabolities, enzymes, agriculture, horticulture, agronomy, bryology, plaleobotany, phytochemistry, enthnobotany, anatomy, ecology, plant breeding, ecology, genetics, chlorophyll, chloroplast, gymnosperms, sporophytes, spores, seed, pollination, pollen, agriculture, horticulture, taxanomy, fungi, molecular biology, biochemistry, bioinfomatics, microbiology, fertilizers, insecticides, pesticides, herbicides, plant growth regulators, medicinal plants, herbal medicines, chemistry, cytogenetics, bryology, ethnobotany, plant pathology, methodolgy, research institutes, scientific journals, companies, farmer, scientists, plant nutrition
Select Language:
 
   
 
 
Can't find? Try Deep Search with ePlantScience.com  
 
Share |
 
   
Main Menu
If navigation gets difficult, please click the main subject or sitemap to get the list of sub-categories
 
 
 
 
 
Related websites
 
 
 
 
 
 
 
 
Section: Genetics » Genetic Engineering and Biotechnology » Recombinant DNA and PCR
 
 
If you like this page, please click:  
 
 
  Construction and screening of genomic and cDNA libraries
 
     
 
Content
⇒ Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA)
⇒ Restriction enzymes in cloning
⇒ Techniques used in recombinant DNA 
⇒ Cloning vectors for recombinant DNA
⇒ Plasmids as vectors
⇒ Bacteriophages as vectors
⇒ Plant and animal viruses as vectors
⇒ Transposons as vectors
⇒ Artificial chromosome vectors for cloning large DNA segments
⇒ Construction of chimeric DNA
⇒ Palindromes and staggered cleavage
⇒ Adding poly dA at the 3' ends of the vector and poly dT at the 3' ends of DNA clone
⇒ Blunt end ligation by T4 DNA ligase
⇒ Cloning in bacteria and eukaryotes
⇒ Cloning in bacteria
⇒ Cloning in eukaryotes
⇒ Molecular probes 
⇒ Labelling of probes
⇒ Applications of molecular probes
⇒ Construction and screening of genomic and cDNA libraries
⇒ Gene amplification : PCR and its applications
⇒ cDNA library from mRNA
⇒ Colony (or plaque) hybridization for screening of libraries
⇒ Gene Amplification : PCR and Its Applications
⇒ The basic polymerase chain reaction (PCR)
⇒ Different schemes of PCR
Construction and Screening of Genomic and cDNA Libraries
In order to isolate one or more related genes from a genome, we like to prepare a mixture of clones each carrying DNA derived either from the genomic DNA or from cDNA (derived from the mRNA isolated from a specific metabolically active tissue of an organism). This mixture may contain thousands of clones, which when derived directly from the genomic DNA are collectively called a genomic library. Similarly when these clones are cDNA, they are collectively called a cDNA library. Construction and use of these libraries will be discussed in this section.

Genomic library by shotgun experiment
Cloning an entire genome in the form of a library of random genomic clones (without identifying them) is often called a shotgun experiment. In this experiment, genomic DNA is extracted, broken into fragments of reasonable size by a restriction endonuclease and then inserted into a cloning vector to generate a population of chimeric vectors (Fig. 39.17). A set of fragments cloned in this manner is called a genomic library. Once such a library is available, then clones can be perpetuated indefinitely in a plasmid vector and retrieved whenever needed for a variety of purposes, mainly for identification and isolation of a gene, whenever a specific probe is available.

Formation of a genomic library using recombinant DNA technique.
Fig. 39.17. Formation of a genomic library using recombinant DNA technique.

Genomic libraries can be prepared by using a number of restriction endonucleases, one at a time, so that fragments of varying sizes having cuts at different places of the genome will be available. However this may lead to cuts at inconvenient places, including sites within a gene, so that fragments having complete genes will be difficult to obtain. In order to overcome this difficulty, we use the following strategy in the shotgun experiment : (i) We use restriction endonucleases, which have short (4bp) recognition sequences, so that such a sequence may be frequently distributed, (ii) Conditions are used which give only partial digests, so that a particular restriction site is only occasionally cleaved, and long fragments without having any breaks on recognition sites available within a gene can be easily obtained. This technique of shotgun experiment leads to the construction of a random genomic library, in which all fragments have same fragment ends thus helping in retrieval of a fragment from the vector with the help of the same enzyme.

The number of fragments representing every sequence of the genome increases with genome size. For instance, for a probability level of 99% that all sequences are present in our library of a species, we may need 1,500 cloned fragments in E. coli; 4,600 in yeast; 48,000 in Drosophila melanogaster and 8,00,000 in a mammal like human being. Libraries reaching these desired limits have been prepared in all these cases.
 
     






     
     
 
 
     
 
Copyrights 2009 © ePlantScience.com