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GENETIC ENGINEERING CHAPTER 20 • Genetic engineering is the ability of humans to modify and manipulate DNA for: – – – – – – – Identification of genetic disorders Gene therapy Crop and food production Tailoring medicines for the cancer Creating recombinant medicines, vaccines Forensics Analyzing evolutionary relationships • In order to do these, scientists need the genes to study and sequence I. DNA Cloning • Involves inserting DNA into a vector and cloning it into a cell for production of multiple copies A. Vectors – DNA molecules that will carry foreign DNA into cells: plasmids, BACs, YACs • Plasmid: small circular DNA found in bacteria. • • Antibiotic resistant gene allows selection of bacteria that took up plasmid • Multiple cloning site allows insertion of foreign DNA • Bacterial artificial chromosome (BAC): large plasmid that can carry large DNA fragments in bacterial cells • Yeast artificial chromosome (YAC): derived from yeast DNA and used to clone really large DNA fragments into eukaryotic cells B. restriction enzymes • found naturally in bacteria to protect them from invading viruses • molecular scissors that cut DNA at specific nucleotide sequences • enzyme binds to DNA at that sequences and cuts between the sugar and phosphate on both strands – cuts can leave blunt or sticky ends Constructing recombinant DNA: II. How to clone a gene using a plasmid vector A. Basic procedure – Cut the DNA of interest and vector with the same restriction enzyme (genomic or cDNA) – Fragments are mixed and ligase seals fragments together – Vector DNA will incorporate the fragments of source DNA in them creating recombinant plasmid – Some vectors will NOT incorporate foreign DNA. They are nonrecombinant – Cells are transformed by mixing vector with cells – Recombinant cells are selected for using agar plates with antibiotics http://highered.mheducation.com/sites/0072556781/student_view0/cha pter14/animation_quiz_1.html B. DNA libraries • Contain fragmented DNA from a particular species stored in a vector ready for cloning – Genomic libraries: made from genomic DNA – cDNA libraries: made from a species’ mRNA using reverse transcriptase http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_ quiz_3.html Constructing a cDNA library: C. Screening for the gene of interest • Use a radioactive probe that will only recognize gene of interest – Transfer some of the bacteria to a piece of filter paper forming a replica – Incubate filter with radioactive piece of DNA complementary to gene of interest – Make an X-ray film. – Use the X-ray film to isolate colony with gene of interest http://sites.sinauer.com/cooper 5e/animation0412.html III. DNA technology A. PCR • Makes many copies of a DNA sample • Uses a three step cycle: • Heating to about 95o C to separate strands • Cooling to anneal primers • Replication using ___ https://highered.mcgrawhill.com/sites/dl/free/0072835125/126997/a nimation38.html B. Electrophoresis and Blotting 1. Electrophoresis • Uses a gel as a molecular sieve to separate nucleic acids or proteins based on size • A current is applied to gel that causes the charged molecules to move thru the gel forming bands based http://bcs.whfreeman.com/thelifewire on size /content/chp16/1602001.html • Bands are visualized with some type of dye 2. Blotting –After electrophoresis, bands are transferred to a filter paper for analysis of a particular fragment (a gene fragment, mRNA, or protein) –Filter paper is incubated with a “probe” to the particular fragment of interest –Southern blot analyzes DNA –Northern blot analyzes mRNA –Western blot analyzes proteins – http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html Northern blot 3. DNA sequencing • Small stretches of DNA are sequenced using dideoxy chain termination method • Uses dideoxynucleotides (ddNTP) mixed with normal ones • Each type of ddNTP has a different fluorescent tag • DNA sequence is http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch read apter15/animation_quiz_1.html Manual sequencing done in 1980s and 1990s Automated sequencing done now: IV. The human genome is polymorphic • Genetically, humans are 99% identical but: – Alleles have small nucleotide differences – Different individuals have different numbers of short tandem repeats: a sequence of 2-5 nucleotides repeated over – This is restriction fragment length polymorphism SNPs: Short Tandem Repeats: A. Creating genetic profiles using STR analysis • There are many regions on chromosomes where STRs exist. • The number of tandem repeats in each region varies from individual • By using PCR on the STR’s of an individual, you can create a genetic profile • • https://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation40.html Cloning an organism: