Chapter 13: DNA Technology 13-1 The New Genetics 13-2 DNA Technology Techniques 13-3 Practical Uses of DNA Technology
13-1 Chromosomes and Inheritance I. Manipulating Genes (base sequences) • Genes must be IDENTIFIED and ISOLATED before INSERTED from one cell type to another.
Critical Thinking (1) List three ways that genetic engineering could be used to improve the lives of humans.
(1) DNA Technology (modern application, Genetic Engineering) • Use of “restriction ENZYMES” and “cloning VECTORS” to change the ACTIVITY of genes (and thus cells).
Critical Thinking (2) What are some common genetic disorders that afflict the human population that may be treatable with DNA biotechnology?
(A) Restriction Enzyme (isolated from bacteria cells) • Cuts DNA into SMALLER pieces at specific SEQUENCES of bases. (Ex: EcoRI recognizes CTTAAG and GAATTC cuts between A -G bases)
(1) Sticky Ends (complementary base-pairs MUST match to bond) • SINGLE chain “TAILS” RESULT when DNA is CUT by enzymes at specific restriction sites.
(B) Cloning Vectors (i.e., recombinant plasmids) • Carriers used to TRANSFER foreign DNA from one organism TO another.
(1) Plasmid (accepts foreign DNA) • RING of bacterial DNA that is ISOLATED and CUT so a DONOR GENE can be inserted.
(2) Donor Gene (collected FOR the genomic library) • Gene ISOLATED from an organism which is then INSERTED into a HOST plasmid gets reinserted into the DIVIDING BACTERIUM.
(3) Gene Clone • As BACTERIAL CELL divides, copies of DONOR GENE are made and PLASMIDS containing these GENE CLONES can be COLLECTED.
II. Transplanting Genes • Plasmids are used to TRANSFER a gene to bacteria SO bacteria will produce a DESIRED PRODUCT.
(1) Insulin • Protein that MOVES glucose INTO cells; (Bacteria are engineered to manufacture MASS quantities of human insulin for DIABETICS).
(A) Isolating a Gene • To isolate insulin GENE, a R.E. is used to CUT the human DNA into pieces (i.e., isolating the insulin gene FROM other genes).
(1) Genomic Library (PROVIDES a source of DONOR genes) • R.E. will CUT an organism’s GENOME into many PIECES ENTIRE SET makes up the GENOMIC LIBRARY.
(B) Producing Recombinant DNA • Inserting a DONOR gene (human insulin gene) into a cloning vector (bacterial plasmid) results in a RECOMBINANT DNA.
(1) Recombinant DNA (two or more sources) • Processed DNA combination from DONOR and HOST source.
(C) Cloning DNA • Recombinant plasmid is COPIED many times, making CLONES of the GENE for INSULIN.
(1) Transgenic Organisms (express foreign DNA, doesn’t originally own) • HOST organism that is recipient of recombinant DNA from ANOTHER source (or even ANOTHER species).
Critical Thinking (3) The FDA does not require special labels for genetically engineered food products that are identical to similar products produced by traditional breeding techniques. Do you think that genetically engineered food products should be labeled as such? Why or why not?
III. Expression of Cloned Genes (challenges have led to TWO techniques) (1) To INDUCE a bacterial host cell to express a FOREIGN gene involves the additional transfer of PROMOTERS that turn on the DONOR gene. (2) The DONOR gene is inserted NEXT to a gene that is normally produced in LARGE quantities within the cell. (i.e., the donor gene is expressed ALONG WITH the host cell’s frequently expressed gene).
13-2 DNA Technology Techniques I. DNA Fingerprint (investigative tool) • PATTERN of BANDS (film) showing FRAGMENTS of an individual’s DNA.
(A) Making a DNA Fingerprint A FOUR-Step Process (1) DNA sample is CUT into many fragments by R.E. (2) DNA fragments are SEPARATED by gel electrophoresis. (3) Radioactive probes ADDED will bind to select DNA fragments. (4) Photographic FILM allows visualization resulting in a DNA fingerprint.
(1) Restriction Fragment Length Polymorphism (RFLP) Analysis (1st) • Taking DNA and CUTTING it into fragments using different R.E. (THESE GET LOADED INTO GEL). NOTE: The NUMBER of fragments and LENGTH of each fragment VARY from person to person (UNIQUE GENETIC IDENTITY).
(2) Gel Electrophoresis (2ndfragments MOVE through a GEL) • SEPARATES fragments BY SIZE (smaller = further DOWN) using LANES and 2 opposite (+/-). NOTE: DNA fragments (-) MOVE towards the POSITIVE (+) end & DISTANCE traveled is dependent on the SIZE.
(3) Radioactive Probes (3rd Selected probes added to DNA AFTER gel ) • Bind to DNA, forming BANDS when exposed to photographic FILM. (RESULTS in DNA fingerprint) NOTE: The DNA fragments on the gel are BLOTTED onto FILTER PAPER once they are done running (TO PRESERVE THE PATTERN).
(B) Accuracy of DNA Fingerprints (MORE variability MORE accuracy) • MOST ACCURATE NONCODING regions where DNA REPEATS OVER AND OVER; found in individual’s genome (called HYPERVARIABLE regions) NOTE STATISTIC: DNA fingerprint compares the HV patterns at FIVE different SITES, and it is HIGHLY unlikely (1 in a million) that ALL five sites compared will MATCH EXACTLY between TWO people.
(C) Polymerase Chain Reaction (PCR allows you to make a DNA fingerprint ) • PCR used to turn a SMALL sample into THOUSANDS of copies of DNA (i.e., the MORE DNA available, the BETTER the fingerprint). • In order to RUN PCR, you must have a SUPPLY of… (1) Original DNA sample (trace amount) (2) DNA Polymerase (DNA Builders) (3) Primers (DNA Starters) (4) DNA Nucleotides (A, T, C, G)
(1) Primer (STARTS replication of trace DNA) • A single-stranded DNA required for INITIATION of DNA replication during PCR.
II. Human Genome Project (46 countries, 1990-2003) 2 shared GOALS have been SET… (1) To figure out the BASE SEQUENCE of entire human genome (approximately 3 billion base pairs—100,000 genes) (2) To map (identify AND isolate) the location of GENES on each of our 46 chromosomes.
The BENEFITS of learning our GENOME may include… (1) Improving diagnoses, treatments, and CURES for ~ 4,000 GENETIC disorders. (2) Improving our understanding of HOW genomes are organized in OTHER species, including how EVOLUTION may occur.
(A) Gene Therapy (early 1990’s experimental treatment) • Introducing (through a vector) a HEALTHLY GENE into a cell OR by CORRECTING a gene defect in a cell’s genome. EX: Nasal sprays (w/ NORMAL cystic fibrosis gene) are INHALED into nose and lungs, where there are cells affected by diseased c.f. genes.