Download DNA Technology

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://media.hhmi.org/hl/10Lect2.html?start=39:49&en
d=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
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