Recombinant DNA Technology……….. BTEC3301
DNA Libraries • How do you identify the gene of interest and clone only the DNA sequence you are interested? Read Pg 63
DNA Libraries Library screening of gene of interest • Libraries are collections of cloned DNA fragments from a particular organisms contained within bacteria or virus as a host.
DNA Libraries Library screening of gene of interest • Libraries can be kept or saved as such for a long time and screened to pick up the gene of interest whenever required.
DNA Libraries Library screening of gene of interest • Two types of libraries which are typically used for cloning are genomic DNA libraries and complimentary DNA libraries cDNA libraries.
DNA Libraries Human genomic DNA libraries • Human DNA is cleaved with a restriction enzyme to several fragments. • These fragments are cloned into plasmids
DNA Libraries Human genomic DNA libraries • Hence a human genomic library consists of a collection of bacteria each containing a different fragment of human DNA.
DNA Libraries Library screening • Once the genomic library or cDNA library is created, it must be screened for gene of interest.
DNA Libraries Library screening • Although libraries are used for cloning and identifying a gene of interest, Polymerase chain reaction (PCR) is a much more rapid approach to cloning than building a library and screening for a gene of interest
Hybridization DNA hybridization • The pairing of two DNA molecules, often from different sources, by hydrogen bonding between complementary nucleotides.
Hybridization DNA hybridization • This technique is frequently used to detect the presence of a specific nucleotide sequence in a DNA sample. • Nucleic acid hybridization on membrane filters is a simple, sensitive, and specific means of detecting nucleic acid sequences of interest.
Hybridization DNA hybridization • Western Blotting is used to analyze proteins which have been immobilized on nitrocellulose/nylon filters.
Hybridization DNA hybridization • Southern blotting (pg75) • Southern Blot (hybridization) was devised by Ed Southern in 1975 for the identification of DNA fragments that are complementary to a known DNA sequence.
The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying small quantities of DNA.
Polymerase Chain Reaction (PCR) Diagrammatic steps in PCR process (Review): • Three major steps in PCR: 1. Template denaturation 2. Primer annealing 3. Primer extension
Go to Animation courtesy of the following websites:References • http://faculty.plattsburgh.edu/donald.slish/PCRmov.html (Animation) • http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html • http://www.people.virginia.edu/~rjh9u/pcranim.html ( PCR Animation) • http://www.escience.ws/b572/L3/L3.htm (PCR Animation) • http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect4.html • http://en.wikipedia.org/wiki/Polymerase_chain_reaction • http://www.escience.ws/b572/L3/L3.htm • http://allserv.rug.ac.be/~avierstr/principles/pcrani.html