Recombinant DNA Technology………..

1. Recombinant DNA Technology……….. BTEC3301

2. DNA Libraries • How do you identify the gene of interest and clone only the DNA sequence you are interested? Read Pg 63

3. 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.

4. 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.

5. 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.

6. DNA Libraries Human genomic DNA libraries • Human DNA is cleaved with a restriction enzyme to several fragments. • These fragments are cloned into plasmids

7. 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.

8. DNA Libraries Library screening • Once the genomic library or cDNA library is created, it must be screened for gene of interest.

9. 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

10. Hybridization DNA hybridization • The pairing of two DNA molecules, often from different sources, by hydrogen bonding between complementary nucleotides.

11. 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.

12. Hybridization DNA hybridization • Western Blotting is used to analyze proteins which have been immobilized on nitrocellulose/nylon filters.

13. 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.

14. The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying small quantities of DNA.

15. Polymerase Chain Reaction (PCR) Diagrammatic steps in PCR process (Review): • Three major steps in PCR: 1. Template denaturation 2. Primer annealing 3. Primer extension

16. Fig. 3.8 The Polymerase Chain Reaction

17. 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