BiotechnologyCh 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 Gene1. 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/olcwe
b/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::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://media.hhmi.org/hl/10Lect2.html?start=39:49&
end=42:08
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 geneshttp://www.youtube.com/watch?v=BBQWWi6cFXU
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-win
ning-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