I. Biology of Viruses & Bacteria (some necessary background as these organisms are important  in biotechnology today)

Viruses   – nonliving, non-cellular particles which infect cells. Viruses consists of: DNA or RNA core surrounded by a Protein Coat.
Viruses multiply by:

• attach to host cell [must have molecular match between virus surface and host cell membrane]
•  the virus or its DNA enters host cell
• viral information (nuclei acids) direct the host cell machinery to synthesize viral proteins and viral DNA/RNA
• viral proteins and viral DNA/RNA are assembled into new viruses
• the new viruses exit the host cell

Retroviruses (=RNA viruses), such as the AIDS Virus (or Human Immunodeficiency Virus, HIV), operate slightly different than the above scenario. Retroviruses have a core of RNA, not DNA. Once inside a host cell viral RNA directs the synthesis of viral DNA (the viral enzyme reverse transcriptase makes this possible).

Bacteria – prokaryotic cells that have a circular DNA chromosome in a convoluted loop and may have smaller loops of DNA called plasmids which carry extra genes.

• accomplish genetic recombination via conjugation (a one-way transfer through a conjugation tube of plasmid DNA between two bacterial cells) and transformation (the uptake of ‘free’ DNA).
• are susceptible to viral attack & restrict the growth of invading viruses with restriction enzymes 
  
• Restriction Enzymes – molecular scissors that cut DNA into fragments; have become essential tools of bioengineering; Different types of restriction enzymes cut DNA at different places based on this: each type of restriction enzyme cuts DNA at a specific location determined by a short sequence of nucleotides, ex. ATCGT may represent a restriction site where a particular restriction enzyme will cut DNA. Wherever ATCGT occurs, DNA is severed by the restriction enzyme. Thus, one long DNA molecule will be cut into different fragments of different lengths depending upon where and in how many locations "ATCGT" is present. Since many restrictions sites are now known to occur just before and after DNA sequences that code for proteins (i.e. genes), carefully selected restriction enzymes are used to snip out genes of choice. Once cut out, a gene can be isolated, cloned, or put into other organisms.

II. DNA Fingerprints
The DNA fingerprint is a column of dark bands on a gel. Click here to see DNA fingerprints on another web site.  Within a column the bands represent different size fragments of DNA, the longer fragments are near the base of the column and the shorter fragments are near the top of the column. Gel electrophoresis is used to separate the DNA in a column and a stain is used to show the presence of DNA within the column. Treated DNA is loaded into one end of a thin gel and an electrical current is passed through the gel causing DNA to move through the gel forming a column of bands (bands further away from the start position contain small fragments of DNA, bands remaining closer to the start position contain larger fragments of DNA).   

Uses of DNA Fingerprints: allows identification of individuals based on DNA evidence, used in:

•  criminology
• conservation biology (monitor grizzly bear roaming)
• to determine degree of kinship (relatedness) between organisms (including humans).
  DEGREE OF "FINGERPRINT" SIMILARITIES REFLECT DEGREE OF KINSHIP
  USED IN GENEALOGICAL STUDIES AND PHYLOGENIC STUDIES (TAXONOMY)

Methods in DNA Fingerprint Production:
PCR - Polymerase Chain Reaction (an automated process developed in 1983)
PCR Requires:

1. Heat resistant form of DNA polymerase (enzyme that promotes DNA synthesis) Heat resistant DNA Polymerase was discovered in the bacterium Thermus aquaticus that lives in hot springs in Yellowstone National Park and this special DNA polymerase is known in the trade as Taq polymerase (“T” for Thermus and “aq” for aquaticus).  

2. Ample supply of nucleotides (A, T, C, & G)

3. Sample of DNA to be cloned.

4. Primers complementary to regions on DNA of interest, viz., primers specific to the regions with tandem repeats (junk) DNA sequences (ex. TTTTC is a tandem segment that is variably repeated in different chromosomes). Primers are single stranded portions of DNA 10-30 nucleotides long [a primer is simply DNA but not double stranded DNA]. Primers are used to locate portions of a genome and prime these for replication. DNA polymerase requires a double stranded portion of DNA as an anchorage point, if you will, in order to begin the  process of building complementary strands alongside exposed single stranded portions of DNA. Primers bind to exposed DNA single strands and allow DNA polymerase to begin DNA replication at that point. Most of the genome will not be replicated, only those portions marked off  by primers.

PCR operates through cycles of heating and cooling. 

[draw tube with piece of DNA to be cloned]

------>[tube w/ twice the DNA] ---------> [tube w/4x DNA] -----> [tube w/8x DNA], … etc.
heat/cool                                    heat/cool                                heat/cool                       

Heating unzips DNA turning all the DNA into single strands.  Cooling allows primers and DNA polymerase to promote base pairing in typical semiconservative replication fashion.  Repeated cycles of heating and cooling produces numerous DNA molecules all identical to source DNA 

III. Methods of Gene Transfer
A. Cross breeding, selective breeding (sexual reproduction)
     An ancient process man still employs

B. Technological Means of inserting DNA into live cells
1. Mechanical injection - micro-needles or micro-pellet guns used to mechanically introduce small amounts of DNA into a cell's nucleus.
2. Use of genetically engineered vectors    
        ex. genetically engineered viruses can carry genes into animal and plant cells.
The cold virus and a herpes virus have been used to as vectors to carry selected genes into human cells; of course these viruses were first modified so as to be incapable of causing harmful infections.
                ex. genetically engineered bacteria can insert genes into plant cells

IV. Applications for Gene Transfers
Advances in biotechnology have made genetic engineering possible. Genetic engineering is the artificial recombination of genes [add a gene (allele) to someone or something that didn’t inherit it naturally]. Genetic engineering produces:
Transgenic organisms - organisms that have had a foreign gene inserted into them.
Why produce transgenic organisms? Below are some of the beneficial applications of genetic engineering.

1. Gene therapy - the transfer of normal genes/alleles into tissues of one w/genetic disorder = “a partial cure” Examples of human genetic disorders currently or soon to be treated with gene therapy: Cystic fibrosis - lung tissue given gene that thins mucus (this gene therapy has had limited success). v Immune disorders (e.g. SCID, "Bubble Boy") - white blood cells given gene that empowers them to arm the immune system. 
Sickle cell anemia - Bone marrow stem cells divide to produce blood cells. To cure one with sickle cell anemia bone marrow stem cells need to be genetically corrected, i.e., only bone marrow cells need to receive the normal hemoglobin gene. Bioengineers recently claimed they  have cured sickle cell anemia in mice in this way and that a cure for sickle cell anemia will be forthcoming in three years.

2. Medical research ex. sickle cell anemia gene put into mice, up until then sickle cell anemia was known only in humans and experimental trials were limited due to the lack of animals on which to experiment. Form more about sickle cell anemia and mice see: http://www.nlm.nih.gov/medlineplus/sicklecellanemia.html

3. Xenotransplantation - transplant animal organs into humans, ex. pig hearts to be transplanted into humans have been genetically engineered so that they will not be rejected by human recipients. Concern: new diseases may infect humans (disease agents previously could not cross from animal to human but xenotransplantation may overcome such barriers).

4. Mass produce human proteins needed to treat human disorders. ex. gene for human blood clotting factor put into pigs, transgenic pigs (genetically engineered pigs) then produce a human blood clotting protein. ex. gene for human insulin put into bacteria, transgenic bacteria then produce human insulin. ex. gene for human growth hormone put in mice (the growth hormone extracted from mice urine)

5. Improve Livestock and Crops – ex. Roundup Ready cotton and Bt corn (see below for explanation of Bt corn and check out this web site from the University of Kentucky).

V.  Concerns for Bioengineering

• Eugenics - genetic enhancement of human race - read quote from 400 BC, Plato’s The Republic “The best of both sexes ought to be brought together as often as possible, the worst as seldom as possible…if our herd is to reach the highest perfection.” See also the novel Brave New World - 1932 by Aldous Huxley- fictional account of a eugenic society made possible by genetic engineering. 
• Genes may escape from cultivated plants and enter wild populations through hybridization between cultivated plants and their wild relatives. Ex., Bt gene [“Bt” is short for the name of the bacterium, Bacillus thuringiensis, from which the gene was originally isolated] has been  inserted into corn and potatoes. The Bt gene codes for the production of an insecticidal chemical specific against butterfly and moth caterpillars. A caterpillar must eat part of the genetically engineered plant to obtain the toxin and then die. Should this gene enter natural populations of related grasses or member of the potato family, unforeseen repercussions may result.
• Bacteria in your intestinal track may(?) pick up potentially harmful genes from genetically engineered food you ingest. The concern is that such bacteria may become pathogenic.