Recombinant DNA Technology……….. BTEC3301. DNA Libraries How do you identify the gene of interest and clone only the DNA sequence you are interested? Read

Recombinant DNA Recombinant DNA Technology………..Technology………..

BTEC3301BTEC3301

DNA LibrariesDNA Libraries

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


Library screening of Library screening of genegene of interest of interest

•Libraries are collections of Libraries are collections of cloned DNA fragments from a cloned DNA fragments from a particular organisms particular organisms contained within bacteria or contained within bacteria or virus as a host.virus as a host.



• Libraries can be kept or Libraries can be kept or saved as such for a long saved as such for a long time and screened to pick time and screened to pick up the gene of interest up the gene of interest whenever required.whenever required.



• Two types of libraries which are Two types of libraries which are typically used for cloning are typically used for cloning are genomic DNA libraries and genomic DNA libraries and complimentary DNA libraries complimentary DNA libraries cDNA librariescDNA libraries..


Human genomic DNA Human genomic DNA librarieslibraries

• Human DNA is cleaved with a Human DNA is cleaved with a restriction enzyme to several restriction enzyme to several fragmentsfragments..

• These fragments are cloned into These fragments are cloned into plasmidsplasmids


Human genomic DNA Human genomic DNA librarieslibraries

• Hence a human genomic library Hence a human genomic library consists of a collection of consists of a collection of bacteria each containing a bacteria each containing a different fragment of human different fragment of human DNADNA..


Library screeningLibrary screening

• Once the genomic library or Once the genomic library or cDNA library is created, it cDNA library is created, it must be screened for gene must be screened for gene of interest.of interest.


Library screeningLibrary screening

• Although libraries are used for Although libraries are used for cloning and identifying a gene of cloning and identifying a gene of interest, Polymerase chain interest, Polymerase chain reaction (PCR) is a much more reaction (PCR) is a much more rapid approach to cloning than rapid approach to cloning than building a library and screening building a library and screening for a gene of interestfor a gene of interest

HybridizationHybridization

DNA hybridization DNA hybridization

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



• This technique is frequently used to This technique is frequently used to detect the presence of a specific detect the presence of a specific nucleotide sequence in a DNA sample.nucleotide sequence in a DNA sample.

• Nucleic acid hybridization on Nucleic acid hybridization on membrane filters is a simple, membrane filters is a simple, sensitive, and specific means of sensitive, and specific means of detecting nucleic acid sequences of detecting nucleic acid sequences of interest.interest.



• Western BlottingWestern Blotting is used to is used to analyze analyze proteinsproteins which have which have been immobilized on been immobilized on nitrocellulose/nylon filters. nitrocellulose/nylon filters.



• Southern blotting (pg75)Southern blotting (pg75)

• Southern Blot (hybridization) Southern Blot (hybridization) was devised by Ed Southern in was devised by Ed Southern in 1975 for the identification of 1975 for the identification of DNA fragments that are DNA fragments that are complementary to a known DNA complementary to a known DNA sequence.sequence.

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

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Diagrammatic steps in PCR process Diagrammatic steps in PCR process (Review):(Review):

• Three major steps in PCR:Three major steps in PCR:

1.1. Template denaturation Template denaturation 2.2. Primer annealing Primer annealing 3.3. Primer extension Primer extension

Fig. 3.8 The Polymerase Chain Fig. 3.8 The Polymerase Chain ReactionReaction

Go to Animation courtesy of the Go to Animation courtesy of the following websites:following websites:

References References http://faculty.plattsburgh.edu/donald.slish/PCRmov.html (Animation) (Animation) http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.htmlhttp://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html http://www.people.virginia.edu/~rjh9u/pcranim.html ( PCR Animation)http://www.people.virginia.edu/~rjh9u/pcranim.html ( PCR Animation) http://www.escience.ws/b572/L3/L3.htm (PCR Animation)http://www.escience.ws/b572/L3/L3.htm (PCR Animation) http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect4.htmlhttp://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect4.html http://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Polymerase_chain_reaction http://www.escience.ws/b572/L3/L3.htmhttp://www.escience.ws/b572/L3/L3.htm http://allserv.rug.ac.be/~avierstr/principles/pcrani.htmlhttp://allserv.rug.ac.be/~avierstr/principles/pcrani.html

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Recombinant DNA Technology……….. BTEC3301. DNA Libraries How do you identify the gene of interest and clone only the DNA sequence you are interested? Read