Chapter 13 Gene Technology

DNA Technology DNA Technology is the manipulation of DNA for practical purposes such as: Identification using DNA fingerprinting Improving food crops Identifying genetic diseases before symptoms appear Research for cures or treatments of genetic diseases

DNA Identification About 0.1% of human DNA differs from person to person. This variation allows us to: Identify people Determine paternity Identify human remains Tracing human origins Provide evidence in criminal cases

Noncoding DNA 98% of our DNA does not code for any protein It is called noncoding DNA It contains many length polymorphisms (variations in the length of the DNA molecule between known genes) It also contains short, repeating sequences known as variable number tandem repeats (VNTR) Geneticists use VNTR’s to determine how rare a particular DNA profile is.

Steps in DNA Identification Isolate the DNA (make copies if needed) Cut DNA into shorter fragments that contain known VNTR areas Sort the DNA by size Compare the size fragments in the unknown sample of DNA to those of known samples of DNA

Copying DNA: PCR Polymerase Chain Reaction (PCR) is a technique that quickly produces many copies of a DNA fragment (it is used if the DNA fragment is very small) Steps: Start with a fragment of DNA with the sequence to be copied Heat to denature and separate the DNA strands Add primers (artificially made pieces of single-stranded DNA that are complementary to the ends of the DNA fragment); DNA polymerase; and nucleotides Primers will bind to original strands; DNA polymerase will add free nucleotides to complete the strands Repeat about 30 times and generate millions of copies

Cutting DNA Restriction Enzymes: bacterial enzymes that recognize specific short DNA sequences and cut them. Cuts are usually asymmetrical and produce “sticky ends” so that other pieces of DNA with complementary sequences can bind to them.

Sorting DNA by Size Gel Electrophoresis —technique that separates nucleic acids or proteins according to their size and charge Steps: Cut DNA with restriction enzymes Place DNA into wells in a thick gel Run electric current through the gel Negatively charged DNA moves towards positive end of the current Smaller fragments move faster and farther Transfer DNA to a nylon membrane and add radioactive probes Expose x-ray film to radiolabeled membrane to produce a DNA fingerprint

Comparing DNA At least 13 VNTR loci comparisons are needed to make a positive DNA identification. Thirteen identical loci make the odds that two people will share a DNA profile about 1 in 100 billion

Recombinant DNA Genetic engineering —the process of altering the genetic material of cells or organisms to allow them to make new substances. It often involves recombinant DNA Recombinant DNA —DNA from one organisms is added to another Pig expressing a jellyfish gene

Cloning Vectors Clone —an exact copy of a DNA segment, a whole cell, or a complete organism: or to make a genetic duplicate Vectors are used to clone DNA fragments They carry foreign DNA from one organism to another They include bacteriophages and plasmids

Plasmids Plasmids —small rings of DNA found naturally in bacterial cells. They replicate separately from bacterial DNA

Plasmids serve as excellent vectors for DNA Steps: Isolate the plasmid from a bacteria cell and the DNA of interest from a human cell (ex: gene coding for insulin) Used restriction enzymes to cut the DNA and the plasmid Mix the DNA and the plasmid together; sticky ends will bond Use DNA ligase to join them by forming permanent covalent bonds Transfer the recombinant plasmids back into bacterial cells Allow bacteria to reproduce (copying the plasmids to the new cells) Use probes to identify the colonies that have the desired gene. These bacterial cells will now be able to produce human insulin

Probes A probe is a strand of RNA or single-stranded DNA that is labeled with a radioactive element or fluorescent dye and that can base-pair to specific DNA, such as the donor gene in recombinant DNA. Probes will glow under UV light, allowing scientists to identify which recombinant colonies have the desired gene.

Medical Applications for DNA Technology Since 1982, more than 30 products made using DNA technology are now on the market. Examples: Human insulin Proteins to treat immune system deficiencies and anemia Clotting factors for people with hemophilia Human growth hormone Interferons for viral infections and cancer Growth factors for treating burns and ulcers

The Human Genome Project The Human Genome Project began in 1990 Its goal was to determine the sequence of all 3.3 billion nucleotides of the human genome and to map the location of every gene on each chromosome. More than 20 labs in 6 countries worked on the project It was completed in 2003 DARN

What Did We Learn? Only 2% of our genome codes for proteins Chromosomes have unequal distribution of nucleotide sequences that are transcribe and translated Our genome is smaller than we thought; only about 30,000 -40,000 genes The same gene can encode different versions of a protein. An organism’s complete set of proteins is called its proteome. Transposons, pieces of DNA that move from one chromosome location to another make up half of our genome and play no role in development The are 8 million single nucleotide polymorphisms (SNP). These are spots where individuals differ by just one nucleotide.

Applications We have discovered the specific genes responsible for many diseases, which can help us to develop treatments and possibly cures for the more than 4,000 human genetic disorders

Genetic Engineering: Medical Applications Gene Therapy —a technique used to treat a genetic disorder by introducing a gene into a patient’s cells. It works best for disorders that result from the loss of a single protein. Ex: Cystic Fibrosis

Cystic Fibrosis patients lack the CFTR gene: Result—excess mucus in lungs Steps of Gene Therapy: Isolate the functional gene from a healthy person Insert it into a viral vector Infect the patient with the recombinant virus (carrying the functional gene) The functional gene temporarily produces the missing protein, improving the symptoms of the disease

Obstacles: Cells that express the highest levels of CFTR are deep in the lungs and are not being reached by the virus Surface cells die off regularly so the treatment must be repeated often Immune reactions to the treatment may occur

Genetic Engineering: Medical Applications Cloning —in 1996, the first clone of an adult mammal was developed. It was a sheep named Dolly. Dolly was created by a process known as cloning by nuclear transfer Steps: Mammary cell with its nucleus was isolated from an adult female sheep Egg cell from another sheep was isolated and the nucleus removed The two cells were fused together and an embryo developed Embryo was transferred into a surrogate mother Dolly was born with nuclear DNA that was identical to the donor of the mammary cell Professor Ian Wilmut and Dolly

Dolly suffered from premature aging and died at the age of 6 Dolly suffered from premature aging and died at the age of 6. However, other cloned animals have not experienced the same problem.

Why Clone Animals? Most animal cloning is done to alter the genome in some useful way. Examples: Altered, cloned goats can secrete human blood clotting factors into their milk. This can then be extracted and used to treat hemophiliacs. Cloned pigs are altered so that their organs can be used in human transplants with a lessened risk of rejection. Altered, cloned mice are used in the study of many human diseases; like cystic fibrosis.

Genetic Engineering: Medical Applications Vaccines —DNA vaccines are now being researched. They are made from the DNA of the pathogen, except the disease-causing genes are removed. When injected into a patient, the patient will mount a defense and build up antibodies. If the real, disease-causing pathogen then enters the body; the antibodies will attack— preventing illness. DNA vaccines to prevent AIDS, malaria and certain cancers are currently being studied.

Genetic Engineering: Agricultural Applications Genetically Modified (GM) Crops are becoming very common. Today, most crops can be genetically engineered to be: More tolerant to environmental conditions Resistant to weed killing herbicides Resistant to insects and other pests Resistant to diseases Improve nutritional value

Ethical Issues Bioethics —the study of ethical issues related to DNA technology. Issues: GM crops: are they healthy for us and are they safe for the environment? Gene therapy: considered unethical if it involves reproductive cells that would affect future generations Cloning: considered unethical to clone human embryos for reproduction Genetic make-up of individuals should remain confidential to reduce the possibility of discrimination.