An introduction...Cell based DNA cloning
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DNA cloning. The desired fragment must be selectively amplified so that it is purified essentially to homogeneity. Thereafter, its structure and function can be comprehensively studied, for example by DNA sequencing, in vitro expression studies, etc., and various manipulations can be achieved to change its structure by in vitro mutagenesis.
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Molecular hybridization. The fragment of interest is not amplified, but instead is specifically detected within a complex mixture of many different sequences. Its chromosomal location can be determined in this way and some information can be gained regarding its structure. If expressed, the sequence of interest can be detected within a complex RNA or cDNA population from specific cells, enabling comprehensive analysis of its expression patterns.
Before DNA cloning, our knowledge of DNA was extremely limited. DNA cloning technology changed all that and revolutionized the study of genetics. Why was DNA cloning such an important technological advance? One important consideration is the tremendous size and complexity of DNA sequences (compared to, say, protein sequences). Individual nuclear DNA molecules contain hundreds of millions of nucleotides. When DNA is isolated from cells using standard methods, these huge molecules are fragmented by shear forces, generating complex mixtures of still very large DNA fragments (typically 50–100 kb long).
Given the above, how can relatively homogeneous DNA populations be prepared from such a complex starting mixture. In the case of DNA from human cells and a wide variety of complex eukaryotic cells, one early approach had been to separate different classes of DNA fragments according to their base composition. The DNA preparation was submitted to ultracentrifugation in equilibrium density gradients (e.g. in CsCl density gradients). When this was achieved, the DNA was fractionated into a major band (the bulk DNA) and several minor satellite DNA bands. The satellite DNA species have different buoyant densities to the bulk DNA because they have unusual sequences and their base composition is different to the majority of the DNA in cells (these properties in turn reflect the involvement of satellite DNA in specific aspects of chromosome structure and function. Although valuable and interesting, the purified satellite DNAs were, however, a minor component of the genome and did not contain genes.
What DNA cloning offered was a general method for studying any DNA sequence.
DNA cloning is a general method of selectively amplifying DNA sequences to generate homogenous DNA populations.
In order to study genes, methods had to be developed to purify them. Because mammalian genomes are complex, any specific gene or DNA fragment of interest normally represents only a tiny fraction of the total DNA in a cell. For example, the β-globin gene comprises only 0.00005% of the 3300 megabases (Mb) of human genomic DNA, and even the massive 2.5 Mb dystrophin gene, the largest gene that has been identified, accounts for only about 0.08% of human genomic DNA.
One way of enriching for gene sequences is to isolate total RNA, or poly(A)+ messenger RNA (mRNA) from suitable cells and convert this to complementary DNA (cDNA) using the enzyme reverse transcriptase. In some cases, this can result in a profound enrichment for specific exonic DNA sequences when the relevant genes are known to be expressed at very high levels in a specific cell type. In most cases, however, the desired gene sequences still represent only a tiny proportion of the total cDNA population.
In order to have a general method of studying a specific DNA sequence within a complex DNA population, the technology of DNA cloning was developed. The essential characteristic of DNA cloning is that the desired DNA fragments must be selectively amplifiedin some way, resulting in a programmed large increase in copy number of selected DNA sequences. In practice, this involves multiple rounds of DNA replication catalyzed by a DNA polymerase acting on one or more types of template DNA molecule. Essentially two different DNA cloning approaches are used:
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Cell-based DNA cloning. This was the first form of DNA cloning to be developed, and is an in vivo cloning method. The first step in this approach involves attaching foreign DNA fragments in vitro to DNA sequences which are capable of independent replication. The recombinant DNA fragments are then transferred into suitable host cells where they can be propagated selectively. In the past, the term DNA cloning has been used exclusively to signify this particular approach.
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Cell-free DNA cloning. The polymerase chain reaction (PCR) is a newer form of DNA cloning which is enzyme mediated and is conducted entirely in vitro. This approach is described in detail in.
Sources:
Content:
https://www.ncbi.nlm.nih.gov/books/NBK7579/
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https://www.ncbi.nlm.nih.gov/books/NBK7579/