Our last lecture dealt with the processes of DNA replication and protein synthesis -- these processes are referred to as the "central dogma" of molecular biology and are the basis for what might be called the "molecular biology revolution" that has been going on for the past 15 years. Hardly a day goes by without something appearing in the paper about a new breakthrough in molecular biology.
Today, we are going to talk about some of the more important new technologies in molecular biology and their applications.
Man has for thousands of years modified the genetic characteristics of organisms for practical purposes -- this has been done through selective breeding (artificial selection)-- breeding of individuals with desired characteristics to produce entire lines of individuals having those characteristics -- e.g. egg-laying in chickens, milk production in dairy cows, increased yields in crop plants such as wheat, rice and corn -- selective breeding has produced many different varieties of agricultural and domestic plants and animals.
Applications of molecular biology have allowed scientists to modify the genetic characteristics of organisms at the DNA level -- the most widely used of these techniques is called recombinant DNA technology -- this technology is revolutionizing agriculture, medicine and forensics.
Recombinant DNA technology -- procedures by which DNA from different species can be isolated, cut and spliced together -- new "recombinant " molecules are then multiplied in quantity in populations of rapidly dividing cells (e.g. bacteria, yeast).
This technology is based on a number of important things:
1. bacteria contain extrachromosomal molecules of DNA called plasmids -- plasmids are circular.
2. bacteria also produce enzymes called restriction endonucleases that cut DNA molecules at specific places -- DNA cut into many smaller pieces called restriction fragments.
Let’s look at the action of restriction endonucleases in a little more detail.
There are many different kinds of restriction endonucleases -- each cuts DNA at a specific site defined by a sequence of bases in the DNA called a recognition site -- several hundred endonucleases have been extracted from bacteria and many are used in recombinant DNA research.
Example: endonuclease called EcoRI cuts DNA where the sequence GAATTC occurs -- GAATTC is the recognition site for this endonuclease -- cut occurs between the G and the A so that the cut is staggered and each fragment has 2 sticky ends.
Diagram on pg. 224:
Cutting with restriction enzymes produces restriction fragments with "sticky ends."
Sticky ends are single stranded ends of the fragment -- can base pair with other sticky ends having complimentary base sequence -- that’s why they are "sticky."
Now, let’s make some recombinant DNA -- insulin is a hormone responsible for regulation of blood sugar levels -- people with defective genes for insulin suffer from diabetes and must take insulin shots.
Before recombinant DNA technology was available, insulin was obtain from animal tissue -- expensive and people frequently had reactions to animal tissue extracts.
Goal of our project is to insert human insulin gene into a bacteria, so the bacteria can "manufacture" insulin for our use -- this is a relatively inexpensive way to make insulin and has already been done.
1. Plasmids are obtained from bacteria cells and cleaved with a restriction endonuclease -- plasmid must contain only a single recognition site so that each plasmid becomes a single fragment with two sticky ends.
2. Human chromosomes (DNA) are collected and cleaved with the same restriction enzyme -- many fragments of chromosomal DNA are formed, but only some of the fragments contain the insulin gene.
3. Chromosomal and plasmid DNA fragments mixed together with enzyme called DNA ligase -- sticky ends of chromosomal fragments weakly bonded (hydrogen bonds) with complimentary bases of plasmid fragments -- circular, recombinant plasmids now formed -- base pairings sealed (covalent bonds) by adding DNA ligase, an enzyme used in normal cells during DNA replication.
4. We now have a collection of recombinant DNA plasmids -- some contain the insulin gene, while others do not -- this collection of plasmids is called a DNA library.
5. Our library is small and must be amplified -- recombinant plasmids are then put back into bacteria, yeast or some other rapidly dividing type of cell -- several ways of doing this -- as these cells divide over and over again, they replicate the plasmids containing the human DNA fragments -- these divisions yield cloned DNA = multiple, identical copies of DNA fragments.
6. Finally, we need to screen our library to identify which bacterial colonies contain recombinant plasmids with the insulin gene -- various ways to do this -- for our project, might be able to do a simple chemical test for the presence/absence of insulin -- discard colonies without the gene and continue growing those that do.
Figure 15.4.
Some miscellaneous points about this procedure:
1. DNA inserted into plasmid may be natural (e.g. human DNA containing gene of interest) or it may be synthetic -- e.g. somatostatin is a protein hormone useful in treating a number of disorders -- it is a 14-amino acid polypeptide -- scientists synthesized a completely artificial stretch of DNA, part of which contained the 52-base sequence coding for somatostatin -- inserted into plasmid and then back into bacteria -- before, almost no somatostatin was available from natural sources -- now bacteria produce it commercially.
2. Plasmids are only one vector for cloning DNA fragments -- scientists have also spliced DNA fragments of interest into the DNA of viruses -- viral vectors then infect rapidly dividing host cells (bacteria, yeast, and other eukaryotic cells) for cloning of DNA.
Many proteins of importance to medical science are now commercially manufactured by biotechnology/pharmaceutical companies using this method -- more economical than previous methods -- besides insulin, others include clotting factor (for hemophilia) and human growth hormone.
Recombinant DNA technology has other medical applications as well -- recall that many human disorders are the result of genes will recessive alleles that produce nonfunctional enzymes -- e.g. PKU, albinism, cystic fibrosis, some types of muscular dystrophy -- we are in the early stages of treating such diseases with gene therapy.
Gene therapy -- supplying patients having defective genes with cells that have nondefective genes -- first federally approved human gene therapy was conducted in 1990.
Young girl suffering from severe combined immune deficiency (SCID) -- caused by a defective gene coding for an enzyme called adenosine deaminase (ADA) -- lack of functional enzyme results in a buildup of toxins that damage lymphoblasts in bone marrow -- lymphoblasts normally give rise to white blood cells, but can’t when damaged -- white blood cells needed for normal immune system function -- people with SCID are unable to generate normal immune response and therefore suffer from a devastating set of infections and disorders.
Scientists obtained white blood cells from patient -- cultured them and inserted normal ADA genes into white cells using recombinant DNA techniques -- put them back in her body -- symptom-free for nearly 5 years with the help of occasional booster treatments.
Here are several other practical applications of recombinant DNA technology:
1. agricultural applications -- genetic engineering is being used to give crop plants genetic characteristics not normally present .
a. strains of wheat cotton, and soybeans carrying a bacterial gene that makes the plants resistant of herbicides used in weed control have been developed.
b. other plants have been engineered for resistance to infection by viruses that spoil fruits (e.g. tomatoes).
c. still others are being engineered to resist attack by insects and this will reduce the need for chemical insecticides.
2. environmental applications.
a. sewage treatment plants rely on microorganisms to degrade organic compounds into less toxic forms -- increasing numbers of compounds are being released that cannot be easily degraded (e.g. chlorinated hydrocarbons) -- scientists are attempting to engineer new microbes that have the ability to degrade these compounds for use in sewage plants.
b. microbes are also being engineered that are capable of detoxifying toxic wastes in spills and waste dumps -- bacterial strains have been developed that can degrade some of the compounds released during oil spills.
Lastly, I want to talk about forensic (legal) applications of recombinant DNA technology -- the O.J. Simpson trial made "DNA fingerprints" a household phrase, yet the general public knows very little about these fingerprints.
In violent crimes, blood or small bits of other tissue may be left at the crime seen and on clothes or other possessions of victims and possible suspects -- in the cases of rape, small amounts of semen can be recovered from the victim.
In the past, forensic scientists could sometimes determine blood and tissue types -- such results were of limited use -- 1) needed fresh samples in amounts sufficient for testing -- 2) because many people in the population have same blood and tissue types, such evidence could only exclude suspects and could be taken as evidence of guilt.
DNA testing overcomes these difficulties: DNA is a hardy molecule and samples don’t have to be fresh; 2) only very small amounts of DNA are needed for testing thanks to a procedure known as PCR; and 3) guilty individuals can be identified with a high degree of certainty since each person (except for identical twins) has a unique DNA base sequence.
Polymerase Chain Reaction (PCR): technique by which a piece of DNA can be amplified very quickly -- billions of copies of a DNA molecule can be made in a few hours
1. DNA put in a test tube and heated to 94oC in a thermal cycler -- a machine that rapidly heats and cools things-- strands of DNA separate exposing nucleotide bases.
2. Free nucleotides and DNA polymerase are supplied and complementary strands are created for the exposed DNA strands -- special type of heat-resistant DNA polymerase is used so it won’t denature at high temperature -- polymerase is called Taq because it is obtained from the Thermus aquaticus, a bacterium that lives in hot springs.
3. Temperature is now lowered and doubled stranded DNA is reformed -- note: all the DNA molecules we started with have replicated so we now have twice as much DNA as we started with.
4. Each cycle of heating and cooling takes about 5 minutes and each cycle doubles the amount of DNA present -- 30 cycles (2.5 hours) will in theory increase the amount of DNA one billion times!!
Figure 15.6.
Using PCR, tiny amounts of DNA from a drop of blood or from the sperm in a drop of semen can be quickly amplified to produce sufficient DNA for testing.
Once a sufficient amount of DNA is available, the forensic scientist is ready to use a procedure called RFLP analysis to generate DNA fingerprints.
1. DNA is first cleaved with one or more restriction endonucleases (remember the specific recognition sites) creating a series of restriction fragments -- fragments will be of differing sizes.
2. Fragments are labeled with a fluorescent dye and then separated by a process called gel electrophoresis -- DNA put in a block of gelatin and exposed to an electrical field -- fragments move according to their size and charge -- big fragments move slower than small ones -- a banding pattern or fingerprint is produced that can be seen.
3. When DNA from different persons are cut into fragments, different banding patterns are observed because base sequences vary slightly from person to person and these sequence differences result in the number and location of endonuclease restriction cuts also being different -- each person therefore will have a unique DNA fingerprint: different numbers and sizes of fragments -- these differences are called restriction fragment length polymorphisms (RFLPs).
In theory, each person (except identical twins) has a unique DNA fingerprint -- in practice, only a small part of the total DNA is tested and the odds of two fingerprints being the same but from different persons is somewhere between 1 in 100,000 and 1 in 1 billion.
Consider the following hypothetical example: semen is recovered from a rape victim -- fingerprints obtained from DNA in sperm cells in sample -- DNA fingerprints also obtained from 3 suspects:
Comparisons of RFLPs indicates semen sample came from Suspect #3.
Next time: History of Evolutionary Thought