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In molecular biology, a library is a collection of molecules in a stable form that represents some aspect of an organism. Two common types of libraries are cDNA libraries (formed from Complementary DNA) and genomic libraries. The nucleotide sequences of interest are preserved as inserts to a plasmid or the genome of a bacteriophage that has been used to infect bacterial cells.
DNA library is a collection of cloned DNA fragments. There are two types of DNA library:
The entire human genome is about 3 x 10 9 bp long while a plamid or F 06 C phage vector may carry
up to 20 kb fragment. This would require 1.5 x 10 5 recombinant plasmids or F 06 C phages. When plating E. coli colonies on a 3" petri dish, the maximum number to allow isolation of individual colonies is about 200 colonies per dish. Thus, at least 700 petri dishes are required to construct a human genomic library. By contrast, as many as 5 x 10 4 F 06 C phage plagues can be screened on
a typical petri dish. This requires only 30 petri dishes to construct a human genomic library.
Another advantage of F 06 C phage vector is that its transformation efficiency is about 1000 times higher than the plasmid vector. cDNA Library A cDNA library represents all of the mRNA present in a particular tissue, which has been converted back to a DNA template by the use of the enzyme reverse transcriptase. It represents the genes that are transcribed in particular tissues under particular physiological, developmental, or environmental conditions. cDNA libraries are useful in reverse genetics, but should not be confused with a genomic library, as it does not represent the entire genome, only a very small (less than 1%) portion which is being transcribed.
Usually a cDNA library is created when reproducing eukaryotic genomic material, whereas genomic libraries are often created when working with genomic target material from bacteria and viruses.
partial digests of genomic DNA are subjected to agarose gel electrophoresis for separation from the mixture of fragments of appropriate size.
Partial digestion as a means of isolating longer, overlapping DNA fragments (Fig: Lodish et al, 4th ed)
Figure: Preparation of the genomic library using F 06 C phage vectors. It is basically the cloning of all DNA fragments representing the entire genome.
A cDNA library is a population of bacterial transformants or phage lysates in which each mRNA isolated from an organism or tissue is represented as its cDNA insertion in a plasmid or a phage vector.
Preparation of cDNA
cDNA Library Construction
cDNA is created from a mature mRNA from an 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) distinguishes mRNA from tRNA and rRNA and can therefore be used as a primer site for reverse transcription
Use of cDNA is absolutely essential when the expression of a eukaryotic gene is required in a prokaryote e.g. a bacterium. This is because eukaryotic genes have introns, which must be removed from their transcripts to yield mature mRNAs. Bacteria do not possess the enzymes necessary for removal of introns. In contrast, functional mRNA molecules do not have introns; hence, their cDNA is also free of introns and can be cloned and expressed in bacteria. For e.g. cDNA for interferon, blood clotting factor VIII C and several other mRNAs have been expressed in bacteria.
cDNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is reduced to remove the large numbers of non-coding regions from the library. cDNA libraries are most useful in reverse genetics where the additional genomic information is of less use.
As previously mentioned, a cDNA library lacks the non-coding and regulatory elements found in genomic DNA. Genomic DNA libraries provide much more detailed information about the organism, but are much more resource-intensive to generate and maintain.
Figure: Screening of a specific DNA fragment. After recombinant F 06 C virions form plaques on the lawn of E. coli , the nitrocellulose filter (membrane) is placed on the surface of the petri dish to pick up F 06 C phages from each plaque.^ Then, the filter is incubated in an alkaline solution to disrupt the virions and release the encapsulated DNA, which is subsequently denatured. Next, the probe is added to hybridize with the target DNA fragment, whose position may be displayed by autoradiography.
Chromosome jumping is a technique of molecular biology that is used as a tool in the physical mapping of genomes. It is related to several other tools used for the same purpose, including chromosome walking.
Chromosome jumping is used to bypass regions difficult to clone, such as those containing repetitive DNA, that cannot be easily mapped by chromosome walking, and is useful in moving along a chromosome rapidly in search of a particular gene.
In chromosome jumping, the DNA of interest is identified, cut into fragments with restriction enzymes, and circularised (the beginning and end of each fragment are joined together to form a circular loop). From a known sequence a primer is designed to sequence across the circularised junction. This primer is used to jump 100 kb-300 kb intervals: a sequence 100 kb away would have come near the known sequence on circularisation. Thus, sequences not reachable by chromosome walking can be sequenced. Chromosome walking can be used from the new jump position (in either direction) to look for gene-like sequences, or additional jumps can be used to progress further along the chromosome.
A technique which produces sets of overlapping DNA clones for studying segments of DNA larger than can be cloned individually. A method for the analysis of large regions of DNA, in which a each end of a large single cloned DNA fragment is used separately to screen recombinant DNA genome library for other clones containing neighbouring sequences.
This method is used to move systematically along a chromosome from a known location and to clone overlapping genomic clones that represent progressively longer parts of a particular chromosome. Chromosome walking is used as a means of finding adjacent genes ( positional cloning ), or parts of a gene which are missing in the original clone as well as to analyse long stretchs of eukaryotic DNA
A small segment of DNA from one end of the genomic clone is used as a probe to isolate clones containing this sequence and adjacent sequences encoding the next portion of the genome. The end sequence of the second clone is used to isolate a third clone and so forth until a series of overlapping clones are isolated. It is necessary to use DNA probes whose sequences are single- copy, otherwise if the probe used is a repeated sequence, then several unrelated recombinants could be identified.
banding pattern, or electropherogram, much like a bar code, that can identify a species or individual (some genes will be vary at the species level and others at the individual level).
The simplest application of this technique is to assess whether a given protein binds to a region of interest within a DNA molecule. The wet lab methodology is summarized, with appropriate selection of reagents discussed, below.
Note: Maxam-Gilbert chemical DNA sequencing can be run alongside the samples on the polyacrylamide gel to allow the prediction of the exact location of ligand binding site.
Labeling
The DNA template can be labeled at the 3' or 5' end, depending on the location of the binding site(s). Labels that can be used are:
Cleavage agent
A variety of cleavage agents can be chosen. Ideally a desirable agent is one that is sequence neutral, easy to use, and is easy to control. Unfortunately none available meet all these all of these standards, so an appropriate agent can be chosen, depending on your DNA sequence and ligand of interest. The following cleavage agents are described in detail:
Advanced Applications
In vivo footprinting
as a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer genomic DNA. There are other ways of mapping features on DNA for longer length DNA molecules, such as mapping by Transduction (Bitner, Kuempel 1981).
One approach in constructing a restriction map of a DNA molecule is to sequence the whole molecule and to run the sequence through a computer program that will find the recognition sites that are present for every restriction enzyme known to man.
Before sequencing was automated, it would have be prohibitively expensive to sequence an entire DNA strand. Even today sequencing is overkill for many applications. To find the relative positions of restriction sites on a plasmid a technique involving single and double restriction digests is used. Based on the sizes of the resultant DNA fragments the positions of the sites can be inferred. Restriction mapping is very useful technique when used for determining the orientation of an insert in a cloning vector, by mapping the position of an off-center restriction site in the insert (Dale, Von Schantz, 2003).
Restriction enzymes:
Restriction enzymes are enzymes that cut DNA at specific recognition sequences called "sites." They probably evolved as a bacterial defense against DNA bacteriophage. DNA invading a bacterial cell defended by these enzymes will be digested into small, non-functional pieces. The name "restriction enzyme" comes from the enzyme's function of restricting access to the cell. A bacterium protects its own DNA from these restriction enzymes by having another enzyme present that modifies these sites by adding a methyl group. For example, E.coli makes the restriction enzyme Eco RI and the methylating enzyme Eco RI methylase. The methylase modifies Eco RI sites in the bacteria's own genome to prevent it from being digested.
Restriction enzymes are endonucleases that recognize specific 4 to 8 base regions of DNA. For example, one restriction enzyme, Eco RI, recognizes the following six base sequence:
5'... G-A-A-T-T-C... 3'
3'... C-T-T-A-A-G... 5'
A piece of DNA incubated with Eco RI in the proper buffer conditions will be cut wherever this sequence appears. As you can see, this site is palindromic; that is, reading the upper strand from 5' to 3' is the same as reading the lower strand from 5' to 3'. As a result, each strand of the DNA can self-anneal and the DNA forms a small cruciform structure:
All restriction enzyme sites are palindromic. This structure may help the enzyme to recognize the sequence that it is designed to cut.
There are hundreds of restriction enzymes that have been isolated and each one recognizes its own specific nucleotide sequence. Sites for each restriction enzyme are distributed randomly throughout a particular DNA stretch. Digestion of DNA by restriction enzymes is very reproducible; every time a specific piece of DNA is cut by a specific enzyme, the same pattern of digestion will occur. Restriction enzymes are commercially available and their use has made manipulating DNA very easy.
Restriction Mapping:
Restriction mapping involves digesting DNA with a series of restriction enzymes and then separating the resultant DNA fragments by agarose gel electrophoresis. The distance between restriction enzyme sites can be determined by the patterns of fragments that are produced by the restriction enzyme digestion. In this way, information about the structure of an unknown piece of DNA can be obtained. An example of how this works is shown below. If we have isolated a clone in pBluescript we know how big the pBluescript portion of the plasmid is (3.0 kilobases) and what restriction enzymes are present in the plasmid. We also know that the insert is 2.0 kb long and that it is inserted the Eco RI site.
At this point, we digest plasmid with an enzyme that we know is in the pBluescript plasmid. For e.g., we know that there is only one Bam HI site in pBluescript, and it is in the multiple cloning site next to the Eco RI site.If we digest this plasmid with Bam HI, there are 2 possibilities: