Recombinant DNA Technology BTEC 3301 DNA Libraries l

Recombinant DNA Technology………. . BTEC 3301

DNA Libraries l 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 c. DNA 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 c. DNA 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 (pg 75) 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. 2. 3. Template denaturation Primer annealing Primer extension

Fig. 3. 8 The Polymerase Chain Reaction

Go to Animation courtesy of the following websites: References l l l l 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/~rjh 9 u/pcranim. html ( PCR Animation) http: //www. escience. ws/b 572/L 3. htm (PCR Animation) http: //homepages. strath. ac. uk/~dfs 99109/BB 211/Recomb. DNAtechlect 4. html http: //en. wikipedia. org/wiki/Polymerase_chain_reaction http: //www. escience. ws/b 572/L 3. htm http: //allserv. rug. ac. be/~avierstr/principles/pcrani. html