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Recombinant Dna Tech

Recombinant Dna Tech

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Published by Arun Sendhil

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Published by: Arun Sendhil on Jun 16, 2012
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Recombinant DNA Technology 
12. Recombinant DNA Technology
 
Key Concepts
 
Recombinant DNA is made by splicing a foreign DNA fragment into a smallreplicating molecule (such as a bacterial plasmid), which will then amplify thatfragment along with itself and result in a molecular clone of the inserted DNA.Restriction enzymes cut DNA at specific target sites, resulting in defined fragmentswith sticky ends suitable for insertion into a vector that has been cut open by the sameenzyme.A collection of DNA clones that encompasses the entire genome of an organism iscalled a genomic library.An individual DNA clone can be selected from a library by using a specific probe for the DNA or its protein product or by its ability to transform a null mutant.DNA fragments of different sizes produced by restriction-enzyme digestion can befractionated because they migrate to different positions on an electrophoretic gel.RNA or restriction-enzyme-cut DNA molecules that have been fractionated by size inan electrophoretic gel can be probed to detect a specific molecule.Restriction-enzyme target sites can be mapped, providing useful markers for DNAmanipulation.A gene can be found by testing overlapping clones radiating outward from a linkedmarker.After a gene has been cloned, its nucleotide sequence can be determined, and thesequence can be used to study gene function and evolution.A pair of replication primers spanning a DNA sequence can be used to amplify thatsequence for isolation.
Injection of foreign DNA into an animal cell.
The microneedle used for injection isshown at right and a cell-holding pipette at left.
(© M. Baret/Rapho/Photo Researchers, Inc.)
Introduction
 
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Recombinant DNA Technology The goal of genetics is to study the structure and function of genes and genomes.Since Mendel's time, genes have been identified by observing standard phenotypicratios in controlled crosses. Clues about gene function first came from correlatingspecific mutations with enzyme and other protein deficiencies. The correlation of mutant sites within a gene with amino acid substitutions in the appropriate protein ledto a better understanding of gene structure and function. To these ideas were addeddiscoveries about the nature of DNA and the genetic code, leading to a fairlycomprehensive understanding of the basic nature of the gene. However, all were
indirect 
inferences about genes; no gene had ever been isolated and its DNA sequenceexamined directly. Indeed, it seemed impossible to isolate an individual gene from thegenome.Although it is relatively easy to isolate DNA from living tissue, DNA in a test tubelooks like a glob of mucus. How could it be possible to isolate a single gene from thistangled mass of DNA threads? Recombinant DNA technology provides thetechniques for doing just that, and today individual genes and other parts of genomesare isolated routinely.Why is gene isolation so important? First, isolation of a gene enables thedetermination of its nucleotide sequence. From this information, the internallandmarks of the gene can be determined—for example, intron number and position.A comparison of DNA sequences between genes also can lead to insights in geneevolution. Converting the DNA sequence of a gene into amino acid sequence by usingthe genetic code leads to comparisons with the protein products of known genes; and,from this knowledge, the function of the gene can often be inferred. Function can also be studied by direct modification of part of the gene's DNA sequence followed by thereintroduction of the gene into the genome. Furthermore, a gene can be moved fromone organism to another. An organism containing a foreign gene is called
transgenic.
 Transgenic organisms can be used either for basic research or for specializedcommercial applications. One application has been to make valuable human gene products such as insulin in transgenic bacteria carrying the appropriate human gene.From this brief overview, we see that gene isolation has become an indispensable toolof modern genetic analysis.What are some examples of interesting genes that could be isolated? The answer depends very much on which biological process is being studied. Let's look at a fewcases. A fungal geneticist studying the cellular pathway for synthesizing tryptophanwould be interested in the genes that, when mutated, confer an auxotrophicrequirement for tryptophan, because each gene would represent a step in the synthetic pathway (seeChapter 10). These genes can be identified through mutation,segregation, and mapping analysis. They would be named
trp1, trp2, trp3,
and soforth. This geneticist would be very interested in isolating and characterizing one or more of these genes. Likewise, human genes that have mutant alleles conferring sometype of functional disorder are interesting for medical and biological reasons. Wehave seen that these genes are identified by pedigree analysis. Two examples covered
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Recombinant DNA Technology inChapters 2and9are the recessive autosomal conditions albinism and alkaptonuria. In these cases, the general nature of the defect has been understood for some time(both are enzyme defects), but it would be very useful to isolate the genes themselves.Other human genes are known from pedigree analysis, but no biochemical function isknown for them. Isolating such genes would be particularly useful because thecharacterization of gene structure might lead to a determination of gene function andthe nature of the disease. A good example is cystic fibrosis, a disease known from pedigree analysis to be caused by an autosomal recessive allele of a gene for which nofunction was known until the gene was isolated and sequenced. Cases such as thesewould be raised in all the organisms used in genetic research.In our consideration of gene isolation, we shall first examine the nature of recombinant DNA and the principle whereby recombinant DNA technology can beused to isolate a gene. Next, we shall examine the methods for isolating specific genessuch as those just discussed.
Making recombinant DNA
 
How does recombinant DNA technology work? The organism under study, which will be used to donate DNA for the analysis, is called the
donor organism.
The basic procedure is to extract and cut up DNA from a donor genome into fragmentscontaining from one to several genes and allow these fragments to insert themselvesindividually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or 
vectors,
for theDNA fragments. The vector molecules with their inserts are calledrecombinant DNA  because they consist of novel combinations of DNA from the donor genome (whichcan be from any organism) with vector DNA from a completely different source(generally a bacterial plasmid or a virus). The recombinant DNA mixture is then usedto transform bacterial cells, and it is common for single recombinant vector moleculesto find their way into individual bacterial cells. Bacterial cells are plated and allowedto grow into colonies. An individual transformed cell with a single recombinantvector will divide into a colony with millions of cells, all carrying the samerecombinant vector. Therefore an individual colony contains a very large populationof identical DNA inserts, and this population is called aDNA clone.A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is inthe bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function. Hence the protocol is often as follows:
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