GENETIC ENGINEERING CHAPTER 20

In order to do these, scientists need the genes to study and sequence 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

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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.mcgraw-hill.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

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III. DNA technology 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.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation38.html

B. Electrophoresis and Blotting 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 on size Bands are visualized with some type of dye http://bcs.whfreeman.com/thelifewire/content/chp16/1602001.html

Southern blot analyzes DNA Northern blot analyzes mRNA 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.mcgraw-hill.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 read http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter15/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: