Lesson 25. RECOMBINANT DNA TECHNOLOGY

Module 6. Bacterial genetics

Lesson 25
RECOMBINANT DNA TECHNOLOGY

25.1 Introduction
Recombinant DNA is a type of DNA that is artificially created by inserting a strand or more of DNA into a different set of DNA. Recombinant DNA is used in genetic modification to create completely new organisms by adding artificial bits or bits of DNA from other organisms to an existing creature. Recombinant DNA is often referred to as rDNA for short.

The technique for making recombinant DNA was first developed in the early-1970s by Herbert Boyer and Stanley Norman Cohen. Their work was built on the work of Daniel Nathans, Hamilton Smith, and Werner Arber, who discovered restriction endonucleases. In 1978 the three were awarded the Nobel Prize for Medicine for this discovery.
Recombinant DNA technology is a technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future. It is an area of medical science that is just beginning to be researched in a concerted effort.

Recombinant DNA technology works by taking DNA from two different sources and combining that DNA into a single molecule. That alone, however, will not do much. Recombinant DNA technology only becomes useful when that artificially-created DNA is reproduced. This is known as DNA cloning (Fig. 25.1).

25.1.1 Here is how recombinant technology works
  • Recombinant technology begins with the isolation of a gene of interest. The gene is then inserted into a vector and cloned. A vector is a piece of DNA that is capable of independent growth; commonly used vectors are bacterial plasmids and viral phages. The gene of interest (foreign DNA) is integrated into the plasmid or phage, and this is referred to as recombinant DNA.
  • Before introducing the vector containing the foreign DNA into host cells to express the protein, it must be cloned. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells.
  • Once the vector is isolated in large quantities, it can be introduced into the desired host cells such as mammalian, yeast, or special bacterial cells. The host cells will then synthesize the foreign protein from the recombinant DNA. When the cells are grown in vast quantities, the foreign or recombinant protein can be isolated and purified in large amounts.

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Fig. 25.1 Recombinant DNA molecule

25.1.2 Other uses for recombinant DNA
Recombinant DNA technology is not only an important tool in scientific research, but it has also impacted the diagnosis and treatment of diseases and genetic disorders in many areas of medicine. It has enabled many advances, including:

25.1.2.1 Isolation of large quantities of protein

In addition to the follicle-stimulating hormone (FSH) used in Follistim® AQ Cartridge (follitropin beta injection) and Follistim® AQ Vial (follitropin beta injection) , insulin, growth hormone and other proteins are now available as recombinant products.

25.1.2.2 Identification of mutations

People may be tested for the presence of mutated proteins that may be associated with breast cancer, retino-blastoma, and neurofibromatosis.

25.1.2.3 Diagnosis of affected and carrier states for hereditary diseases

Tests exist to determine if people are carriers of the cystic fibrosis gene, the Huntington’s disease gene, the Tay-Sachs disease gene, or the Duchenne muscular dystrophy gene.

25.1.2.4 Transferring of genes from one organism to another

People suffering from cystic fibrosis, rheumatoid arthritis, vascular disease, and certain cancers may now benefit from the progress made in gene therapy.

25.1.2.5 Mapping of human genes on chromosomes

Scientists are able to link mutations and disease states to specific sites on chromosomes.

25.2 Main Concepts and Definitions in Recombinant DNA Technology

Making and replicating a desired piece of DNA
  • One little piece of DNA by itself can't be studied. You can't determine its base sequence or its products by looking at it under a microscope.
  • To effectively study DNA, one must manufacture a large quantity of a DNA segment of interest, ‘magnifying’ it for easier study with biochemical methods.
  • To do this, recombinant DNA is made by splicing a DNA fragment of interest into a small DNA molecule (such as a bacterial plasmid) called a vector.
  • Once this is done, one can make huge numbers of the desired DNA fragment by inserting the vector into a very busy piece of DNA in another live cell (such as a bacterium).
  • The bacterium ‘works’ for you by allowing the vector to replicate. As the bacteria multiply, so does DNA.
  • This magnified sample can then be extracted for further study.
A Few Definitions:
  • The organism from which the DNA of interest is extracted is called the donor.
  • The DNA into which the DNA of interest is inserted (often a bacterial plasmid) is called a vector.
  • The organism (or DNA) into which the foreign DNA is inserted is called the recipient.
  • An organism containing an artificially inserted, foreign piece of DNA is said to be transgenic (i.e. the recipient becomes transgenic once the new DNA is inserted).
25.2.1 How is it done?
To excise a piece of DNA from a donor organism, restriction enzymes are used. These act somewhat like ‘enzymatic scissors’, slicing through the DNA at specific, recognized sequences. Once the DNA is excised, DNA ligase is the ‘enzymatic glue’ used to insert it into replicating DNA of the host cell. Note that DNA ligase isn't picky: it can't tell the difference between foreign and host DNA, and this enables the creation of hybrid DNA-DNA from two separate sources (sometimes different species). A vector molecule with an insert of foreign DNA is a recombinant DNA molecule. DNA made from the combined DNA of two (or more) species is sometimes called chimeric DNA after the beast of Greek mythology. Vectors are often mixed with bacterial strains which take them up and incorporate them into their own genomes, a process known as transformation). Vectors may also be replicated autonomously (without being inserted into the bacterial DNA) as the bacterium goes about its daily business. By growing the bacterial strain carrying the desired recombinant DNA vector, one can grow a large number of the desired DNA fragment. This is the DNA clone. Once a large DNA clone (remember: a clone is a group of things, not a single individual) has been grown, the researcher can
  • characterize the DNA (determine its base sequence)
  • make RNA from it
  • make protein from it (after you've made the RNA)
  • modify the DNA to see what happens when it mutates
  • reinsert it into a recipient organism for production of products or further study
25.3 Restriction Enzymes
First discovered in bacteria, restriction enzymes cut DNA at very specific DNA base sequences (called restriction sequences). These enzymes are believed to be a bacterial defense against viruses. Each restriction enzyme recognizes and cleaves a very specific sequence of DNA. Restriction sequences are palindromes: they read the same, forward and backward on the opposite strands. Cutting with restriction enzymes creates highly reactive "sticky ends" that act as attachment points for other fragments of DNA with complementary restriction sequences. By connecting pieces of DNA from two different species (that happen to have the same restriction sites), we create chimeric DNA. Note that restriction sites are a ‘happy accident’ of nature. They have nothing to do with gene function in the organism in which they are found. In fact, they are a defense mechanism, found primarily in bacteria, which function to fragment and destroy the DNA of invading bacteriophages (i.e. ‘bacterium-eating’ viruses) before it can incorporate into the bacterial host's genome to do its dirty work. Bacterial DNA is immune to the bacteria's own restriction enzymes: in its normal state a bacterium's own restriction sites are highly methylated (i.e. the bases have many methyl groups (-CH3 attached), protecting them from the activity of the restriction enzymes.

Restriction enzymes are named for the organism from which they were first isolated. For example
  • EcoRI is isolated from E. coli strain RY13
  • Eco refers to the genus and species (1st letter of genus; 1st two letters of specific epithet)
  • R is the strain of E. coli
  • I (Roman numeral) indicate it was the first enzyme of that type isolated from E. coli RY13.
  • BamHI is isolated from Bacillus amyloliquefaciens strain H
  • Sau3A is isolated from Staphylococcus aureus strain 3A.
Each enzyme recognizes and cuts specific DNA sequences. For example, BamHI recognizes the double stranded sequence:
5'--GGATCC--3'

3'--CCTAGG--5'

25.3.1 Summary
Most restriction enzymes cut only one specific restriction site. Restriction sites are recognized no matter what the DNA's species. The number of cuts in an organism's DNA made by a particular restriction enzyme depends on the number of restriction sites (specific to that restriction enzyme) in that organism's DNA. A fragment of DNA produced by a pair of adjacent cuts is called a restriction fragment. A particular restriction enzyme will typically cut an organism's DNA in to many pieces, from several thousand to more than a million. There is a great deal of variation in restriction sites, even within a species (Everyone in this room has different numbers and locations of restriction sites. Your restriction site numbers and locations are more similar to those of your close family members than to unrelated humans. Although these DNA variations are not phenotypically expressed, the variants can be considered molecular ‘alleles’, and they can be detected with sequencing techniques. This is yet another type of genetic variation of interest to the evolutionary biologist.
Last modified: Tuesday, 9 October 2012, 5:21 AM