Biotechnology Ch 20

20.1 DNA cloning Making copies of segments of DNA Gene cloning – making multiple copies of a gene Why? To make many copies of a gene (amplification) To produce a protein product

Cloning & bacteria Plasmids are frequently used in cloning genes Gene of interest is inserted into plasmid: Recombinant DNA – DNA from 2 different sources Plasmid is inserted into bacteria, bacteria divide, producing copies of genes

Recombinant DNA DNA from two sources is combined http://www.youtube.com/watch?v=8rXizmLjegI

Cloning a Gene 1. Isolate vector and gene of interest Determine vector – molecule that will carry foreign DNA, and gene of interest Vector may have particular genes to aid in recognition of of cell clones vector - bacterial plasmid Has ampR – ampicillin resistance gene Has lacZ gene – catalyzes hydrolysis of lactose sugar – at restriction site, so the enzyme cuts in middle of gene Gene - human gene of interest

Restriction Enzymes Protects bacteria by cutting up foreign DNA Work on specific sequences of DNA, usually symmetrical Result in “sticky ends”

2. Insert gene into vector The same restriction enzyme is used to digest the plasmid (only one recognition site), and human DNA Result- human DNA is cut into many fragments – one is the correct one. Cut plasmids and DNA fragments are mixed together. Sticky ends join through complementary base pairing. DNA ligase is used to form phosphodiester bonds to join DNA molecules.

Making recombinant DNA

3. Introduce cloning vector into cells Bacterial cells take in recombinant plasmids through transformation, taking in DNA from surrounding solution

4. Cloning of cells Bacterial cells are plated out onto nutrient medium with ampicillin and X-gal sugar added Need to determine which bacterial cells contain recombinant plasmids Only bacteria with recomb. plasmids will grow on medium with ampicillin, because of ampR gene Bacteria with the intact lacZ gene turn blue with hydrolysis of X-gal, but bacteria with recomb plasmids cannot process X-gal sugar, so are white

Cloning a gene - video http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::5 35::/sites/dl/free/0072437316/120078/micro10.sw f::Steps%20in%20Cloning%20a%20Gene

5. Identify cell clones with gene of interest Need to find bacteria with plasmids that contain gene of interest, vs. other human DNA fragments Use nucleic acid probe – short strand of DNA or RNA that is complementary to part of gene of interest DNA is denatured, and then radioactive or fluorescent probe is added

Genomic Libraries A collection of many clones A complete set of plasmid-containing cell clones When no single gene is target, genome broken into fragments, each gets recombined into a plasmid Bacterial artificial chromosome – larger than plasmids, hold more genes

Complementary DNA - cDNA Eukaryotic DNA from its original source includes introns To get around this problem, start with a fully processed mRNA strand Use reverse transcriptase to synthesize double stranded DNA Can build a cDNA library

Nucleic Acid Hybridization Can be used to label particular bands of DNA Synthesized radioactively labeled RNA hydrogen bonds with target complementary DNA

Cloning & expressing eukaryotic genes Problems due to differences in how prokaryotic & eukaryotic cells express genes Promoter- use an expression vector with a promoter sequence upstream of insertion site, so host cell recognizes it and will express gene that follows Introns – find processed mRNA, use reverse transcriptase to make complementary DNA (cDNA) that can be used in bacteria

Yeast – hosts for eukaryotic cloning Advantages: Single- celled fungi, easy to grow Have plasmids (unusual for eukaryotes) Eukaryotic host cells can modify proteins after translation, bacteria can’t do this

PCR – Polymerase Chain Reaction Making copies of DNA Uses heating & cooling cycles to: 1) denature – separate DNA strands 2) anneal - bind primers at ends 3) extension -synthesize DNA with DNA polymerase http://www.youtube.com/watch?v=2KoLnIwoZKU

20.2 DNA Technology – analyzing genes Gel Electrophoresis: Use electricity to separate DNA fragments in an agarose gel DNA is negatively charged Longer molecules travel slower than shorter molecules

Restriction fragment analysis DNA can be digested with restriction enzymes, and then analyzed

Genome mapping DNA sequencing – dideoxy chain termination: http://www.youtube.com/watch?v=3JkL_cIRRnw Sequencing by synthesis: http://www.dnatube.com/video/2954/Pyro-Sequencing Human genome sequencing – shot gun sequencing: http://www.youtube.com/watch?v=-gVh3z6MwdU

Analyzing Gene Expression Transcription is a measure of gene expression Use probes to measure amt of mRNA present, as a way to quantify gene expression DNA microarray assays – a grid of single strand DNA fragments, get tested for hybridization with cDNA molecules http://media.hhmi.org/hl/10Lect2.html?start=46:55& end=49:52

FISH Fluorescent in situ hybridization - to determine which cells are expressing certain genes http://www.youtube.com/watch?v=BBQWWi6cFX U

Gene function In vitro mutagenesis Add inactive genes with a marker (mutated genes), put the gene back into the cell so it “knocks out” the normal functioning gene RNAi – use of synthetic double strand mRNA to breakdown mRNA or block translation; acts to knock out certain genes

Knock out mice – Mario Capecchi http://on.aol.com/video/nobel-prize-winning- scientist-on-knockout-mice-517890437 RNAi – use of double stranded mRNA molecules to “knock out” genes

SNP – Single nucleotide polymorphism A single base pair site where variation is found Used as genetic markers for particular diseases Find common genetic marker for people who are affected with a disease Study nearby region of DNA to look for genes involved in disease

Cloning – organisms from single cells Plants – cells from adult plants incubated in medium can grow into normal adult plants Animals – nuclear transplantation (i.e. Dolly) The adult cells need to be dedifferentiated Nucleus from a differentiated adult cell is transplanted into a egg cell with the nucleus removed Problems – defects: premature death, obesity, liver failure Problems appear to be due to chromatin methylation issues

Stem Cells Stem cells are unspecialized and can differentiate into specialized cells. In a stem cell, DNA is arranged loosely. In a differentiated cell, genes not needed are shut down

Embryonic stem cells – from embryos in the blastula stage Can reproduce indefinitely, can differentiate into many different cell types – pluripotent Why are they valuable? have the potential to supply cells to repair damaged or diseased organs Adult stem cells – can differentiate, but not as widely as embryonic stem cells

Induced Pluripotency - Shinya Yamanaka took adult fibroblast cells (connective tissue cells) Reprogrammed the cells to become pluripotent- to being capable of differentiating into different cell types (like stem cells) https://www.youtube.com/watch?v=i-QSurQWZo0 Reprogrammed cells with master transcription factors Yamanaka won the Nobel Prize in Medicine 2012

Current research with pluripotency Problems with traditional genetics approach due to cancer causing genes Use of small compounds to mimic transcription factors Use of drug like chemicals to enhance reprogramming

Another way to create pluripotent cells Haruko Obokata from Riken Center for Developmental Biology, Kobe, Japan Stimulus-triggered acquisition of pluripotency (STAP) Took lymphocytes from mice, bathing them in acid solution for about 30 minutes. Cultivated the cells by adding a special protein. In two to three days, the process had transformed the cells into pluripotent cells. They developed into nerve and muscle cells.

Mouse embryo injected with pluripotent cells (labeled with fluorescent protein)

Applications of DNA Technology Diagnosis of diseases Gene Therapy Production of proteins for market Other pharmaceutical products Forensic evidence Environmental Cleanup Agricultural applications