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Molecular Biology Sabah Linjawi ١
Lecture 14
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Molecular Biology Sabah Linjawi ٢
Genomic Libraries
A genomic library is a population of host bacteria
Each of which carries a DNA molecule that was inserted into a cloning vector
Such that the collection of cloned DNA molecules represents the entire genome of the source
organism.
This term also represents the collection of all of the vector molecules
Each carrying a piece of the chromosomal DNA of the organism
prior to the insertion of these molecules into the host cells.
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Molecular Biology Sabah Linjawi ٣
Types of Vectors
1- Plasmids: Have a capacity of 10 kb.
2- Phages: Have a capacity of 23 kb.
3- Cosmids: Have a capacity of 45 kb.
4- Yeast artificial chromosomes (YAC): Have a capacity of 1000 kb.
DNA library is a collection of cloned DNA fragments.
There are two types of DNA library:
The genomic library contains DNA fragments representing the entire genome of an organism.
The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell.
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Molecular Biology Sabah Linjawi ٤
Genomic library
Preparation of the genomic library using λ phage vectors.
It is basically the cloning of all DNA
fragments representing the entire genome.
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Creating a library
The DNA molecules of an organism of interest are isolated.
The DNA molecules are then partially digested by an endonuclease restriction enzyme.
Sometimes, the DNA molecules are digested for different lengths of time in order to ensure that all the DNA has been digested to manageable sizes.
The digested DNA molecules are separated by size using agarose electrophoresis
And a suitable range of lengths of DNA pieces are isolated and ligated into vectors.
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Molecular Biology Sabah Linjawi ٦
Creating a library
The vectors can then be taken up by suitable hosts.
The hosts are kept in liquid media and can be frozen at -80°C for a long period of time.
Usually the hosts are bacteria that do not contain any plasmids, so as to be sensitive to antibiotics.
The process of subdividing genomic DNA into
clonable elements and inserting them into hosts is called creating a library, a clone bank or a gene bank.
A complete library of host cells will contain all of the genomic DNA of the source organism
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Molecular Biology Sabah Linjawi ٧
Making a genomic library in bacteriophage lambda vector
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Molecular Biology Sabah Linjawi ٨
Size of library
A genomic library is a set of clones that together represents the entire genome of a given organism.
The number of clones that constitute a genomic library depends on:
(1) the size of the genome in question
(2) the insert size tolerated by the particular cloning vector system.
For most practical purposes, the tissue source of the genomic DNA is unimportant because each cell of the body contains virtually identical DNA (with some exceptions
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Molecular Biology Sabah Linjawi ٩
Applications of genomic libraries
Determining the complete genome sequence of a given organism
Serving as a source of genomic sequence for generation of transgenic animals through
genetic engineering
Study of the function of regulatory sequences in vitro
Study of genetic mutations in cancer tissues
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Molecular Biology Sabah Linjawi ١٠
cDNA libraries
A cDNA library is a collection of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells
Which together constitute some portion of the transcriptome of the organism.
cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism.
Similarly, tissue specific cDNA libraries can be produced.
In eukaryotic cells the mature mRNA is already spliced
Hence the cDNA produced lacks introns and can be readily expressed in a bacterial cell.
While information in cDNA libraries is a powerful and useful tool since gene products are easily identified
The libraries lack information about enhancers, introns, and other regulatory elements found in a genomic DNA library
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cDNA Library Construction
cDNA is created from a mature mRNA from a
eukaryotic cell with the use of an enzyme known as reverse transcriptase.
In eukaryotes, a poly-(A) tail (consisting of a long sequence of adenine nucleotides)
Which distinguishes mRNA from tRNA and
rRNA and can therefore be used as a primer site
for reverse transcription
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The procedure of RNA processing for
protein genes
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mRNA extraction
Firstly, the mRNA is obtained and purified from the rest of the RNAs.
Several methods exist for purifying RNA such as trizol extraction and column purification.
Column purification is done by using oligomeric dT nucleotide coated resins where only the mRNA having the poly-A tail will bind.
The rest of the RNAs are eluted out.
The mRNA is eluted by using eluting buffer and some heat to separate the mRNA strands from oligo-dT.
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Formation of a cDNA library
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Molecular Biology Sabah Linjawi ١٥
cDNA construction
Once mRNA is purified, oligo-dT (a short sequence of deoxy-thymine nucleotides) is tagged as a complementary primer which binds to the poly-A tail providing a free 3'-OH end
The mRNA is removed by using a RNase enzyme leaving a single stranded cDNA (sscDNA).
This sscDNA is converted into a double stranded DNA with the help of DNA polymerase.
However, for DNA polymerase to synthesize a complementary strand a free 3'-OH end is needed.
This is provided by the sscDNA itself by generating a hair pin loop at the 3' end by coiling on itself. The polymerase extends the 3'-OH end.
Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids.
The cloned bacteria are then selected , commonly through the use of antibiotic selection.
Once selected, stocks of the bacteria are created which can later be grown and sequenced to compile the cDNA library
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Molecular Biology Sabah Linjawi ١٦
Genomic library & cDNA library
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Applications of cDNA libraries
Discovery of novel genes
Cloning of full-length cDNA molecules for in vitro study of gene function
Study of the repertoire of mRNAs expressed in different cells or tissues
Study of alternative splicing in different cells or
tissues
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Molecular Biology Sabah Linjawi ١٨
References
Molecular Biology. P.C. Turner, A.G. Mclennan, A.D.
Bates & M.R.H. White.School of Biological
Sciences, University of Liverpool, Liverpool, UK.
Second edition. BIOS Scientific Publishers, 2000.
en.wikipedia.org/wiki/Library_(biology)
en.wikipedia.org/wiki/Genomic_library
www.web-books.com/MoBio/Free/Ch9B.htm
homepages.strath.ac.uk/~dfs99109/BB211/Lod7-1
www.le.ac.uk/.../images/genomiclibrary_000.jpg
