Introduction

Advances in microbiology techniques have made a profound impact on the course of the development of genetic engineering. This section will examine several of these techniques, including their development, application, and impact in the field. It is more technical than the other sections of this project.

Brief Background History
In the 1920's, English physician Frederick Griffith found evidence of some unknown "transforming principle" that turned an innocuous strain of the virus Streptococcus pneumoniae into a virulent, deadly form in mice. It was generally believed that this principle was either some sort of nucleic acid or protein.

In 1944, Oswald Avery, Colin MacLeod, and MacLyn McCarty showed convincing evidence that deoxyribonucleic acid (DNA) was this transforming principle, although the scientific community was not convinced of this until a 1952 paper published by Alfred D. Hersey and Martha Chase, dealing with bacteriophage DNA.

In 1950, Columbia's Erwin Chargaff reported quantitative correlations between the nucleotides of DNA (the purine adenine with pyramidine thymine; purine guanine with pyramidine cytosine), which were eventually found to represent the bonding characteristics of the DNA nucleotides.

In 1953, Francis Crick and James Watson at Cambridge University proposed a double-helical, antiparallel model of the DNA structure that is generally accepted today, although variations of this model exist.(Purves 242-5)

The Structure of DNA

Figure 1. The structure and bonding characteristics in the Watson-and-Crick model of DNA configuration. (from Purves, 247.)

Figure 2. The "central dogma" of biology. Describes DNA replication as a means of propagation; transcription as a means of converting DNA to RNA information; translation as a means of converting RNA information into amino acid polypeptides, which are proteins. (from Purves, 258)

To understand the majority of the concepts presented in this section, it is necessary to understand the basic structure of DNA under the Watson and Crick double-helix model. A single DNA nucleotide consists of three parts: a sugar molecule, deoxyribose; any one of four nitrogenous bases (adenine(A), cytosine(C), guanine(G), and thymine(T) in DNA); and a phosphate group. Nucleotides are linked by phosphodiether bonds; that is, the phosphate group of one nucleotide binds with the sugar molecule of the next nucleotide. The phosphate groups link the 3' carbon of deoxyribose in one nucleotide to the 5' carbon of the next nucleotide. This notation is useful in describing the direction of DNA; each DNA strand is said to have a 5' "head" followed by a 3' "tail."

A large number of these nucleotides are linked together to form long strands of DNA nucleotides; a DNA molecule has two strands, running 5'-3' in opposite directions. These two strands are held together through the base-pairing of the nitrogenous bases; as Chargaff observed, there is a correlation between adenine-thymine and cytosine-guanine. These bonds were determined to be hydrogen bonds; two hydrogen bonds hold together A-T bonds and three hydrogen bonds hold together C-G bonds.
Another important nucleic acid is ribonucleic acid (RNA), which is similar to DNA in most aspects. However, the sugar molecule in RNA is ribose, and in general, RNA consists of only one strand. Furthermore, in RNA, the thymine nitrogenous base is replaced by uracil(U), which is similar to thymine and shares the same bonding characteristics.(Purves, 245-7)

Eventually, the central dogma of biology was developed to explain the role of DNA, which states that "a given gene is transcribed to produce a messenger RNA[mRNA] complementary to one of the DNA strands, and that transfer RNA[tRNA] molecules translate the sequence of bases in the mRNA into the appropriate sequence of amino acids."(Purves, 258)

These groundbreaking revelations on the structure and nature of DNA were vital developments in spawning the biotechnology industry.

Restriction Enzymes
Figure 3. Cloning an insert into plasmid, and the transformation of bacteria with the engineered recombinant plasmid. (from Watson, 74)
A nuclease is "an enzyme that breaks down nucleic acids into their constituent nucleotides by cleaving their chemical bonds."(Access Excellence glossary, http://www.gene.com/ae) In 1970, the first restriction enzyme, HindII, was discovered, after researchers at the Johns Hopkins University discovered that foreign DNA from bacteriophage (a virus that infects bacteria) were broken down by Haemophilus influenzae. Restriction enzymes are nucleases which cut the phosphodiester bonds linking nucleic acids at specific sequences in a DNA molecule. (Watson, 64-5) For instance, the Johns Hopkins University group discovered that HindII cleaves DNA in the center(between the third and fourth basepairs) of a six-base pair sequence, which, because of its sequence specificity, is known as the HindII "recognition sequence":
5' G T T or C | A or G A C 3'
3' C A A or G | T or C T G 5'
HindII is an example of a blunt-end restriction enzyme. Another type of restriction enzyme is the "sticky-end" nuclease which cleaves asymmetrically, leaving 3' and 5' protruding ends. Sticky-end nucleases are far more important in genetic engineering than blunt-end nucleases. An example of this is EcoR1:
5' G | A A T T C 3'
3' C T T A A | G 5'

These sticky ends tend to bond with complementary sequences of bases. The discovery of these types of enzymes led to the birth of the recombinant DNA industry.
(From Restriction Enzymes- Background, Genentech Acess Excellence, http://www.gene.com/ae/AE/AEC/CC/restriction.html)
Once DNA (often in the form of a plasmid, which is a small circular ring of DNA which exists separately from a cell's main DNA) is cut with a sticky-end producing restriction enzyme, it is possible for a genetic engineer to clone in foreign DNA with the appropriate complementary basepairs at both ends. An enzyme known as DNA ligase permanently joins the fragments. (Watson, 70-71, 74).
5' G AATT foreign DNA AATT C 3'
3' C TTAA sequence TTAA G 5'

Green: plasmid DNA, Blue: foreign DNA

In 1973, Herbert Boyer and Stanley Cohen at Stanford and the University of California at San Francisco used the Escherichia coli plasmid pSC101, which contains a single EcoR1 recognition site, to create hybrid plasmids with foreign DNA fragments possessing cohesive ends generated by EcoR1 and DNA ligase. (Watson, 73-74.)

In January 1979, the National Institutes of Health eased restrictions on recombinant DNA experimentation imposed in July 1976, and the cloning of viral cancer genes became permissible, bringing about the creation of a new biotechnology industry.(Watson, 75-76).

Once a suitable plasmid has been completed, it can be inserted into host cells-- a process known as transformation if the host cell is a bacterium, or transfection if the host cell is eukaryotic. (Purves 315) There are various methods of accomplishing this; for instance, for transfection of eukaryotic cells, a process known as electroporation can be used. Electroporation entails placing cells in a solution containing DNA, and applying a brief electrical impulse, which causes temporary holes to develop in the cell membranes. (Watson, 221-2) Often, for bacterial transformation, plasmids have antibiotic resistance genes as a selection mechanism to destroy plasmid-less cells; because transformation is an imprecise science, putting transformed bacteria into cultures with antibiotics will kill those cells which did not, for whatever reason, intake the plasmid. (Purves 317)

Figure 4. Agarose gel electrophoresis, twelve lanes. Stained with ethidium bromide, exposure under UV light. This common technique is used to create restriction maps of restriction enzyme recognition sites in plasmids.(from Invitrogen 1997 catalog, 99)

Gel Electrophoresis
Agarose Gel Electrophoresis
Figure 5. The molecular weight markers in agarose gel electrophoresis. Each band is a specific length of DNA. This marker would be loaded in a lane on an agarose gel, and would be used to estimate the size of a DNA fragment from on the same gel.(from Watson, 66)

A vital tool in DNA research is agarose gel electrophoresis, which determines the relative size of restriction fragments. This technique utilizes the negative charge of DNA molecules. Dilutions of agarose form gels through which DNA will migrate when induced with an electric field; larger fragments of DNA move at slower rates than smaller fragments of DNA as a result of the size of the molecule and the viscosity of agarose. Using a staining substance such as ethidium bromide, which binds to DNA and fluoresces under UV light, it is possible to determine the location and relative intensity of a DNA signal. (Watson, 66)
Agarose gel electrophoresis is vital in creating restriction maps of endonuclease recognition sites in plasmids, as well as in several DNA-sequencing techniques. The first restriction map was created by Johns Hopkins researcher Daniel Nathans, who produced a map of the sites on plasmid SV40 using HindII.
Polyacrylamide Gel Electrophoresis
Another important type of electrophoresis is polyacrylamide gel electrophoresis (PAGE). Polyacrylamide gels operate on the same principles as agarose gel electrophoresis, though it is used mainly for protein analysis. Because of protein secondary structure (the three-dimensional structure of proteins), the protein must be loaded into the gel with a solution to linearize the protein. Most protein gels utilize a solution of sodium dodecyl sulfate(SDS) which breaks apart noncovalent bonds in proteins, as well as mercaptoethanol or a similar substance to reduce disulfide bonds. As in agarose gels, shorter proteins will migrate more rapidly through the gel, and after staining (Coomassie blue), bands can be detected.(Stryer, 47)
Figure 6. Diagram of polyacrylamide gel electrophoresis(PAGE) aparatus. The solution containing the material to be separated is loaded from the top (using the pipettetip seen in diagram). The actual gel is situated vertically between glass plates, and the aparatus is emerged in electrophoresis buffer. (from Stryer, 47)
Various other assays depend on electrophoresis. The most common are Western blotting, for studying proteins; Southern blotting, for studying genomic DNA, and Northern blotting, for RNA analysis. (Watson, 129f)

Restriction Fragment Length Polymorphisms
Using restriction enzymes on DNA, the DNA molecule will be cut into smaller fragments depending on its sequence and the enzyme used. Mutations in DNA can eliminate or add restriction enzyme recognition sites, or change the size of the DNA fragments. Using a specific probe to identify DNA position, restriction enzymes, and gel electrophoresis, it is possible to find the restriction fragment length polymorphisms(RFLP, pronounced rifflip) exhibited by an individual or characteristic of a disease.(Purves, 340)

Figure 7. A common gel-based method for sequencing DNA. (from Watson, 68)

DNA Sequencing
Determining the sequence of DNA was a vital step in increasing knowledge about the organization of DNA, and of genes and chromosomes. Early sequencing efforts yielded the nucleotide coding of the relatively small tRNA molecules, which are about 75-80 nucleotides in length. Until the discovery of specific restriction enzymes, it became apparent that any plans to effectively decipher the nucleotide sequence of large DNA molecules would be hopeless. (Watson 63-4) The discovery of specific restriction enzymes and the subsequent advances in sequencing that followed were major developments in genetics.

Techniques
There are a number of different methods of sequencing DNA. However, many of them use electrophoresis gels. These methods usually employ DNA nucleotides lacking an oxygen atom (named ddNTP's) which are less reactive than regular DNA nucleotides (dNTP's) and will stop nucleotide elongation if they are added to the chain. These techniques usually involve adding dNTP's, ddNTP's, primers, and DNA polymerases along with the unknown DNA's. The ddNTP's can either be labeled with dyes, or four separate mixtures, each containing a different ddNTP, could be run in four separate lanes on a gel. The lengths of the fragments on a gel will indicate the sequence of a DNA fragment. (Purves, 326-7, Watson, 69).

Human Genome Project
Recognizing the importance of knowing the complete DNA sequence of the human genome, an ambitious sequencing effort is underway. Known as the Human Genome Project, this effort was begun in the United States in 1990, and is expected to last fifteen years. The project, which is coordinated by the U.S. Department of Energy and the National Institutes of Health, aims to identify all the estimated 80,000 genes in the human genome, as well as determining the approximately 3 billion basepairs of the human genome. The project passed its halfway point in April, 1998. (Human Genome Project Information, www.ornl.gov/TechResources/Human_Genome/home.html)
The human genome project has spurred the development of new sequencing technologies. For instance, the "first-generation gel-based sequencing technologies" described above have been used to sequence specific regions of interest in the genome. This method has been automated. Still, it is an inefficient and expensive method; the best available method can sequence only 50,000 to 100,000 basepairs per year at a cost of $1 to $2 per base. As a result, a major focus of the project has dealt with developing new automated sequencing methods.

Second-generation technologies will increase speed and accuracy by an order of magnitude, while simultaneously lowering the cost per base of sequencing. These methods include "high-voltage capillary and ultrathin electrophoresis to increase fragment separation rate" (in other words, improving the gel electrophoresis method) and the "use of resonance ionization spectroscopy to detect stable isotope labels." Third-generation sequencing technologies are expected to have no gel electrophoresis involvement. (Mapping and Sequencing the Human Genome: Primer on Molecular Genetics,http://www.ornl.gov/hgmis/publicat/primer/prim2.html)

Polymerase Chain Reaction
Need
Analysis of specific genes is a complex task since a microbiologist must isolate a particular gene in a complex genome that can contain an immense number of genes. There are a number of techniques in molecular genetics which deal with this isolation problem. However, many of these techniques are immensely time-consuming since they involve cloning and detecting specific sequences of DNA. In the mid-1980s, Kary Mullis described a technique known as polymerase chain reaction(PCR), which greatly simplified this task. (Watson, 79).

Method
PCR is primarily a method of DNA amplification. It can easily produce billions of copies of a single DNA target sequence in a very short period of time.(Purves 326) PCR uses the natural processes of DNA replication, as well as DNA polymerase. The process initiates by heating the DNA double helix so that the hydrogen bonds holding the two strands together are broken; the individual DNA strands are known as the DNA templates. A primer-- a small section of double-stranded DNA used to initiate DNA polymerization-- is added to the solution, in addition to DNA nucleotides. The primer and the DNA nucleotides will bind to the single-stranded templates, and DNA polymerase will be used to permanently bond the new single-strands of DNA.
The process is repeated any number of times, though a complication arises because the primers cannot anneal to the templates, and thus the new strands cannot be polymerized, at the high temperatures required to separate the DNA strands. Therefore, in any PCR cycle, the temperature of the solution must vary between 55 to 95 degrees C. Rather than adding polymerase at every cycle, which would be a tedious and time-consuming task, PCR is accomplished using the DNA polymerase from a thermophilic bacteria Thermus aquaticus which operates best at around 75 degrees C. This DNA polymerase is commonly known as Taq polymerase in honor of the bacterium.(Access Excellence, Polyermase Chain Reaction- Xeroxing DNA, www.gene.com/ae/AB/IE/PCR_Xeroxing_DNA.html) Taq polymerase is stable even at 94 C, so PCR is simplified by adding the polymerase at the beginning of PCR, rather than after each step as was the case when E. coli polymerase was used. Besides simplicity, Taq polymerase has also improved the specificity and sensitivity of the method. The higher operating temperatures allowed by using the Taq polymerase as opposed to the E. coli polymerase means that the chance of the primer incorrectly binding to an incorrect part of the DNA template is greatly reduced. (Watson 84)

Application
There are a variety of applications for PCR. PCR is a DNA-specific method, so the DNA to be amplified does not have to be particularly well-purified. Furthermore, because of the inherent stability of DNA, and because of the low volumes of template DNA required to initiate PCR, PCR has been used to amplify DNA from extraordinary sources. For instance, human papilloma virus DNA has been found in cervical carcinoma biopsies embedded in paraffin for over 40 years. PCR can be used to amplify DNA from dried blood samples. PCR has even been used to amplify DNA from Egyptian mummies. DNA amplified by PCR can be used like any other DNA, and is very useful for sequencing.(Watson, 85-6)
PCR can be used in a number of medical applications as well. Using special primers, for instance, it is possible to screen for sickle-cell anemia. Other variations of this technique include using radioactive primers. PCR is invaluable in medical applications involving minute blood samples.(Watson, 552-554)

Conclusions
In 1921, Frederick Banting and Charles Best at the University of Toronto treated diabetic patients with the protein insulin, extracted from pancreatic tissue. (Watson 812) In 1928, Alexander Fleming accidentally discovered the antibiotic penicillin. (Converging on DNA, Access Excellence, http://www.gene.com/ae/AB/BC/1900-1953.html) However, with the development of new recombinant DNA technologies, new medical breakthroughs can be "engineered" more easily and with more specificity than in the past.
References
Purves, William K., Gordon H. Orians, and H. Craig Heller. Life: The Science of Biology, 4th ed. Sunderland, Massachusetts: Sinauer Associates, 1995.
Stryer, Lubert. Biochemistry, 4th Ed. New York: W.H. Freeman and Company, 1995.
Watson, James, and Michael Gilman, Jan Witkowski, and Mark Zoller. Recombinant DNA, 2nd Edition. New York: W.H. Freeman and Company, 1992.
Last updated on