Cloning Reproductive Cloning Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly – Died at age 6 suffering from lung cancer and crippling arthritis (Dorset sheep normally live years) Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.
Cloning cont. Therapeutic Cloning Therapeutic cloning, also called "embryo cloning," is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. In November 2001, scientists from Advanced Cell Technologies (ACT), a biotechnology company in Massachusetts, announced that they had cloned the first human embryos for the purpose of advancing therapeutic research. To do this, they collected eggs from women's ovaries and then removed the genetic material from these eggs. A skin cell was inserted inside the enucleated egg to serve as a new nucleus. The egg began to divide after it was stimulated with a chemical. The results were limited in success. Although this process was carried out with eight eggs, only three began dividing, and only one was able to divide into six cells before stopping.
Recombinant DNA Def: DNA in which genes from 2 different sources are linked Genetic engineering: direct manipulation of genes for practical purposes Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products
Bacterial plasmids in gene cloning
Gene Cloning Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific Restriction site: recognition sequence for a particular restriction enzyme Restriction fragments: segments of DNA cut by restriction enzymes in a reproducable way Sticky end: short extensions of restriction fragments DNA ligase: enzyme that can join the sticky ends of DNA fragments Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)
DNA Analysis & Genomics PCR (polymerase chain reaction) Gel electrophoresis DNA Fingerprint DNA sequencing Human genome project
Polymerase chain reaction (PCR) Amplification of any piece of DNA without cells (in vitro) Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers Applications: fossils, forensics, prenatal diagnosis, etc. ent/anisamples/molecularbiology/pcr. html ent/anisamples/molecularbiology/pcr. html rad.cnpg.com/lsca/videos/ScientistsF orBetterPCR/ rad.cnpg.com/lsca/videos/ScientistsF orBetterPCR/
DNA Analysis Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length y/gelelectrophoresis.html
Agarose gel electrophoresis
Larger DNA Fragments Smaller DNA Fragments Negative end (-) Positive end (+)
After staining with ethidium bromide to visualize DNA… Large DNA pieces Small DNA pieces - pole + pole
DNA Sequencing Determination of nucleotide sequences (Sanger method, sequencing machine) Genomics: the study of genomes based on DNA sequences Human Genome Project
Practical DNA Technology Uses Diagnosis of disease Human gene therapy Pharmaceutical products (vaccines) Forensics Animal husbandry (transgenic organisms) Genetic engineering in plants Ethical concerns?
What is the current status of gene therapy research? The Food and Drug Administration (FDA) has not yet approved any human gene therapy product for sale. Current gene therapy is experimental and has not proven very successful in clinical trials. Little progress has been made since the first gene therapy clinical trial began in In 1999, gene therapy suffered a major setback with the death of 18-year-old Jesse Gelsinger. Jesse was participating in a gene therapy trial for ornithine transcarboxylase deficiency (OTCD). He died from multiple organ failures 4 days after starting the treatment. His death is believed to have been triggered by a severe immune response to the adenovirus carrier. Another major blow came in January 2003, when the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. FDA took this action after it learned that a second child treated in a French gene therapy trial had developed a leukemia-like condition. Both this child and another who had developed a similar condition in August 2002 had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID), also known as "bubble baby syndrome." FDA's Biological Response Modifiers Advisory Committee (BRMAC) met at the end of February 2003 to discuss possible measures that could allow a number of retroviral gene therapy trials for treatment of life-threatening diseases to proceed with appropriate safeguards. In April of 2003 the FDA eased the ban on gene therapy trials using retroviral vectors in blood stem cells.
What factors have kept gene therapy from becoming an effective treatment for genetic disease? Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy. Immune response - Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients. Problems with viral vectors - Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease. Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy. For more information on different types of genetic disease,
DNA Fingerprinting Involves several techniques to analyze and compare DNA from separate sources DNA is taken from one or more of these sources, chemically cut into segments and the segments are sorted by length in a gel using electrophoresis Segments contain specific sequences of repeating base pairs that are variable from person to person (VNTR) Segments are tagged radioactively and form a visual pattern on X-ray film These DNA fingerprints can be compared to DNA found as evidence, DNA of suspects and DNA of victims
The Basics of the Procedure DNA is extracted A chemical process called polymerase chain reaction (PCR) uses enzymes to amplify the amount of DNA Sections of DNA where there are repeats are cut in order to determine the number of repeats present The fragments are put on an electric field that sorts them by size (gel electrophoresis) The fragments are then placed onto a nylon membrane where they are treated with radioactive probes
The probes stick to some DNA fragments but not to others, due to complimentary base pairing A piece of X-ray film is put on top and a spot is produced on the film where the probes stick Using a ruler, scientists measure the position of the spots on the film and produce a set of numbers The odds of two individuals having the same pattern are any where from thousands to one to trillions to one, depending on what type of analysis is used.
VNTR Specifics In the US, 13 STR (single tandem repeats) loci have been selected for forensic typing and inclusion in CODIS (Combined DNA Index System). The average random match probability when all 13 are typed is less than 1 in a trillion among unrelated individuals!!! Because each of the loci used in forensic DNA typing is on a different chromosome, they are each inherited independently of one another. This allows forensic scientists to calculate the frequency of any given DNA profile by multiplying each individual allele frequency together.
Downfalls of DNA DNA is useful, but there are some downfalls: Contamination of evidence is possible – can occur in lab through the air Transfer of Evidence from pieces of evidence is also possible because uneducated crime scene processors often mix pieces of evidence that may contain DNA together in the same package (coffee cups, doorknobs, telephones, etc.) Testing not always conclusive – need to use more loci when creating a DNA fingerprint.