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MBY364 Theme 4 summary
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Full summary of theme 4 (genome and cDNA libraries) made using lecture notes, the textbook and class notes.
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CLONING STRATEGIES: GENOME AND cDNALIBRARIES
LIBRARIES
- Library - collection of cloned DNA fragments representative of the entire study
population
- These must be screened for the cloned gene of interest
- There are mainly two types of libraries depending on the source of DNA used
- Genomic library - fragments of total genomic DNA cloned into a
suitable vector
- cDNA library - reverse transcribed mRNA (cDNA) cloned into a
suitable vector
- Both are a collection of recombinant clones
- Genomic library is larger and stays constant as the DNA content of the cell
remains the same
- cDNA library is smaller and contains only the coding regions and what is
expressed
- It is representative of the mRNA expressed only under certain conditions
therefore differences occur between cells types/tissues and life stages
- cDNA sequences reveal expression profiles in different cell types,
developmental stages and in response to natural or experimentally stimulated
external stimuli
- These sequences provide useful information about splice isoforms and their
abundance in different tissues and developmental stages
- Isolating sequences from libraries
- Divide the source DNA into manageable fragments and clone
everything
- This entire collection of clones is known as a library
- Screen the library to identify the clone of interest
- Alternatively a sequence can be identified by amplifying the target sequence
using PCR and then cloning the individual fragment
GENOMIC LIBRARIES
- A representative genome library contains all of the DNA sequences in the
entire genome of an organisms
- An insufficient number of clones will give rise to an incomplete genome library
and therefore lack genes or parts of genes - not representative
- Need to determine the number of recombinant clones (n) required to construct
the library
- n = genome size/fragment size
- E.g. the human haploid genome has a size of 2.8 x 10 6kb
, - EcoRI (6bp recognition sequence) is used to cut the DNA to generate
4kb fragments
- n = (2.8 x 106kb)/4kb = 7 x 105 independent recombinant clones
- This calculates how many recombinants must be prepared and screened in
order to have a reasonable chance of including the desired sequence
- Problems with constructing a library using this strategy:
- The gene may be cut internally, especially large genes when we want
to obtain the whole gene fragment
- Fragments may be too short to include the flanking regions or whole
gene clusters
- The fragment may be larger than the vector can accept therefore
cannot be cloned
- Solutions to these problems:
- Cloning of random fragments of a large size (+- 20kb)
- This results in no systematic exclusion of any sequence
- The clones will overlap each other and the sequence of very large
genes can be assembled
- Larger fragments have fewer clones needed for a complete library
- The number of clones required to create a representative library will be
- n = (2.8 x 106kb)/20kb = 1.4 x 105 independent recombinant clones
- In reality more than this number of clones will be needed as sampling
variation will lead to the inclusion of some sequences several times and
exclusion of other sequences
- Therefore it is necessary to calculate the number of independent recombinant
clones (N) needed to obtain a particular probability for a target sequence
- N = ln(1-P)/ln(1-1/n)
- n = the number of recombinants obtained
- P = the desired probability of finding the gene of interest
- E.g. for a 95% probability, with insert size of 20kb, what is the number of
independent clones required in a human genome library?
- n = (2.8 x 106kb)/20kb = 1.4 x 105 independent clones
- N = ln(1 - 0.95)/ln(1 - 1/1.4 x 105) = 4.2 x 105 recombinant clones
needed
- This ensures 95% probability of finding a particular gene in the library
HOW TO GENERATE APPROPRIATELY SIZED FRAGMENTS
- Mechanical shearing can be used
- Commonly used procedure involved REs
- Target DNA is digested with either one or a mixture of two REs that have
tetranucleotide recognition sites
- Therefore can occur frequently in the target DNA
- Perform a partial digest resulting in many of the fragments being of large size
- Partial digest - reaction isn't allowed to run to completion
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