DNA Technology

Genetic Engineering Genetic engineering is the direct manipulation of genes for practical purposes Manufacturing protein products Biotechnology is the manipulation of organisms or their components to make useful products Selective breeding of cattle Making wine and cheese Genetic engineering/biotechnology are major areas of research in biology today

DNA Cloning Preparing identical copies of a specific gene Can be used to produce a protein product (gene  protein) Copies of a gene can be used for: Basic research Endowing an organism with a new metabolic capability pGLO

Practical Applications of Gene Cloning Gene for pest resistance inserted into plants Genes used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth Others??

Restriction Enzymes & Recombinant DNA enzymes that cut DNA molecules at specific locations Look for specific sequences: Ie: ATTACCGTA Recombinant DNA – DNA in which nucleotide sequences from 2 different sources (often different species) are combined into the same DNA molecule

Recombinant DNA

Recombinant DNA Sticky Ends: Single-stranded ends of a restriction fragment (piece of DNA) Sticky ends of restriction fragments can form hydrogen-bonded base pairs with other sticky ends These bonds are sealed with DNA ligase

Plasmids A plasmid is a DNA molecule that is not part of the chromosome in bacteria Often give bacteria an “advantage” such as antibiotic resistance Plasmids are often used as cloning vectors Take foreign DNA into a cell and replicate there

Polymerase Chain Reaction (PCR) PCR is a process used to copy a segment of one or many DNA molecules quickly in vitro (in a test tube) PCR can make BILLIONS of copies of a target segment of DNA in a few hours

Polymerase Chain Reaction (PCR) Denaturation Mixture is heated to separate the DNA strands 2. Annealing Mixture is cooled to allow primers to form (beginning of DNA replication) 3. Extension DNA polymerase adds nucleotides to 3’ end of primer

Polymerase Chain Reaction (PCR)

Applications of Polymerase Chain Reaction (PCR) Used to amplify (copy) DNA from a variety of sources Fragments of DNA from a 40,000-year-old woolly mammoth DNA from fingerprints or tiny amounts of blood, tissue, semen at crime scenes… If there is not much DNA available for analysis, PCR can produce enough DNA for other applications (electrophoresis, etc.)

Restriction Fragment Analysis Restriction fragment analysis detects certain differences in the nucleotide sequences of DNA molecules

Restriction Fragment Length Polymorphisms (RFLPs) RFLPs are differences in restriction sites on homologous chromosomes that result in different restriction fragment patterns Everyone has a different DNA sequence, so the restriction enzymes will cut the DNA at different places The length of the restriction fragments can be analyzed using electrophoresis

Gel Electrophoresis The DNA fragments produced by restriction enzymes are sorted by gel electrophoresis Sorted by rate of movement through a gel in an electric field Distance is inversely proportional to the length of the fragment – Long fragments don’t go far, short ones do The banding pattern (where the fragments stop) is used to analyze/identify DNA

Gel Electrophoresis

The Human Genome Project Largely completed in 2003 Essentially, a detailed map of the entire human genome Genetic mapping – Relative order of genes (1st, 2nd, 3rd, etc.) Physical mapping – Definitive distance between genes (2457 bp, etc.) DNA sequencing – complete nucleotide sequence (ATCCG…)

Applications of DNA Technology Medical Applications Diagnosing genetic diseases Amplifying (cloning) genes and analyzing them Gene therapy Altering the genes of an afflicted individual Replacing a “defective” allele with a “normal” allele To be effective, it must be done in cells that multiply throughout life Bone marrow cells (somatic cells) Gametic cells (sperm/egg) Lots of ethical issues involved here!

Applications of DNA Technology Pharmaceutical Products Using DNA cloning, genes that code for the production of specific proteins can be made in vitro Human insulin (diabetes) Human growth hormone (HGH) Vaccines

Forensic Evidence DNA fingerprinting (RFLP analysis and electrophoresis) is used to solve crimes The likelihood that 2 people would have the same DNA fingerprint (restriction enzymes would cut their DNA at all the exact same places and therefore have the same banding pattern) is between 1 in 100,000 and 1 in 1,000,000,000 Also used in paternity testing

Applications of DNA Technology Environmental Cleanup Many bacteria can extract heavy metals (copper, lead, nickel) from their environments These genes could be added to other organisms to clean up mining wastes Bacterial strains can degrade some of the chemicals released during oil spills

Ethics & DNA Technology The progress in genetic technology has raised new ethical questions: Who should have the right to examine someone else’s genes? How should that information be used? Should a person’s genome be a factor in suitability for a job or eligibility for insurance?