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Chapter 16Lecture and
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In 2002, this kitten named “Carbon Copy” was produced by reproductive cloning – the first cloned pet
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Chapter 16
Genetic Technology
Gene Cloning
Genomics: Techniques for Studying Genomes
Biotechnology
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Chapter Outline:
Recombinant DNA technology Use of laboratory techniques to isolate and
manipulate fragments of DNA
Recombinant DNA contains DNA from two or more sources
Once inside a host cell, recombinant molecules are replicated to produce identical copies or clones
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Gene Cloning
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Gene cloning
Creates many copies of a particular gene
To make large amounts of a specific gene’s DNA For DNA sequencing To probe for a similar gene in another species
Or to produce the protein encoded by the gene Purify protein to study structure and function Introduce into bacteria or livestock for pharmaceuticals Introduce into plants and animals to change traits Use for gene therapy to treat disease
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BIOLOGY PRINCIPLE
Biology is an experimental science
The technique of gene cloning allows researchers to study genes and gene products in greater detail.
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Gene Cloning – Step 1
Vector DNA is a carrier for the DNA segment to be cloned
When a vector is introduced into a living cell,it can replicate, making many copies
Common vectors are plasmid or viral vectors
Also need the gene of interest from some organism’s chromosomal DNA
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Gene Cloning – Step 2
To insert chromosomal DNA into vector...
Cut DNA using restriction enzymes Also called restriction endonucleases Made by bacteria as protection against bacteriophages Cut at specific known restriction sites Most restriction sites palindromic May produce sticky ends
DNA ligase must be used to permanently link DNA
Creates a recombinant vector with gene inserted Will also get empty recircularized vector without insert
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Gene Cloning – Step 3
Goal is to have recombinant vector taken up by bacteria Some will take up a single plasmid Most cells fail to take up a plasmid at all
Vector carries a selectable marker Adding antibiotics selects for cells expressing
antibiotic resistance gene, ampR
Gene ampR codes for beta-lactamase that degrades ampicillin, which normally kills bacteria
Growth on ampicillin plates indicates that bacteria contain plasmid with the selectable marker
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Only the cells with the plasmid grow on plates treated with ampicillin
To eliminate recircularized empty vectors, lacZ geneis built into vector Insertion of cloned DNA disrupts lacZ gene lacZ codes for beta-galactosidase, which cleaves
colorless X-Gal into a blue dye Recircularized plasmids will form blue colonies The desired recombinant vectors will form white colonies
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DNA library
Treatment of chromosomal DNA with restriction enzymes yields tens of thousands of different fragments
DNA library – collection of many recombinant vectors each with a fragment of chromosomal DNA
Two types of common DNA libraries Genomic library – inserts derived from chromosomal DNA cDNA library – uses reverse transcriptase to make
complementary DNA (cDNA) from mRNA Lacks introns, so simpler to use
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Electrophoresis
Technique used to separate macromolecules on a gel Can be used to separate DNA or proteins
Can be used to separate molecules based on their charge, size/length, and mass
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Polymerase chain reaction (PCR)
Allows us to amplify DNA (make many copies) No vectors or host cells needed
Ingredients: Primers that match sequences at each end of the DNA fragment dNTPs (deoxynucleotide triphosphates) Taq polymerase – a heat-stable form of DNA polymerase called
DNA run through repeated cycles of denaturation, annealing and synthesis Thermocycler machine automates this process
After 30 cycles of amplification, a DNA sample will increase 230-fold
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Genomics – the molecular analysis of the entire genome (all the DNA) of a species
Two phases Mapping of genome Functional genomics
How do specific DNA sequences impact structure and function of cells and the organism?
Expression analysis – which genes turn on or off in particular cells
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Genomics: Techniques for Studying Genomes
DNA sequencing
Determines base sequence of DNA
Dideoxy chain-termination method Dideoxynucleoside triphosphates (ddNTPs) are missing
the 3’–OH group and cause chain to terminate 4 tubes with copies of the single stranded DNA, each with
a different labelled nucleotide DNA polymerase will make complementary strand until
ddNTP inserted and chain terminates After electrophoresis, DNA sequence can be read by
determining which base is at the end of the DNA strand
Procedure has been automated using fluorescently labeled ddNTPs all in one tube 26
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3′
Recombinant vector
Annealingsite
5′
Primer
Many copies of the recombinant vector, primer,dNTPs, fluorescently labeled dideoxynucleotides(ddNTPs), and DNA polymerase are mixedtogether. Incubate to allow the synthesis of DNA.
CACCGTAAGGACTddGCACCGTAAGGACddTCACCGTAAGGAddCCACCGTAAGGddACACCGTAAGddGCACCGTAAddGCACCGTAddACACCGTddACACCGddTCACCddGCACddCCAddCCddAddC
Separate newly made strands bygel electrophoresis.
981 2 3 4 5 6 7
1011121314
GT G G C A T T C
C
TG
AC
Sequence to be analyzed(target DNA)
Synthesis of short DNA strandsuntil a ddNTP is added.
GTCAGGAATGCCAC
Sequencededucedfrom gel
(a) Dideoxy DNA sequencing method
Fluorescencedetector
C A C C G T A A G G A C T G
(b) Output from automated sequencing
Longest DNAstrand
Laserbeam
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A microarray can identify which genes are transcribed by a cell
DNA microarray or gene chip
Monitors expression of thousands of genes simultaneously
Short sequences of known genes attached to spots on slide
Goal is to find out which genes are transcribed into mRNAin a particular sample of cells mRNA from those cells is used to make fluorescently labeled cDNA
cDNAs that are complementary to the DNAs in the microarray will hybridize
High fluorescence intensity in a spot indicates high levels of cDNA in the sample
Technologies that involve the use of living organisms to benefit humans
12,000 years ago – domestication of livestock Now – molecular genetics
Important medicines are now produced by recombinant organisms Tissue plasminogen activator – for blood clots Factor VIII – for hemophilia Insulin – for diabetes
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Biotechnology
Insulin
In 1982, the U.S. FDA approved sale of human insulin made by recombinant bacteria
Prior to 1982, insulin was isolated from cattle Some people developed allergies and had to use cadaver insulin
Insulin composed of two polypeptides – A and B A and B coding sequence inserted into E.coli Fusion proteins extracted and β-galactosidase removed Purified A and B chain mixed to form functional protein
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BIOLOGY PRINCIPLE
Biology affects our society
This method of making insulin, which was developed by researchers, is now used to make insulin for most
people with insulin-dependent diabetes.
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Gene replacement and knockout
Used to understand gene function and human disease
A transgenic organism carries genes introduced using molecular techniques Also called Genetically Modified Organism (GMO)
Gene replacement – cloned gene recombines with normal gene on a chromosome Only 1 of 2 copies replaced, creating heterozygote Heterozygotes can be crossed to yield homozygotes
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Gene knockout – if cloned gene is a mutation that inactivates function, homozygote will not have gene function
Very useful for studying human disease Used to create models to study disease Used to test effect of therapies
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Molecular pharming
Production of medically important proteins in livestock mammary glands
Certain proteins are more likely to function when expressed in mammals Post-translational modification Degraded or improperly folded in bacteria High yield in cows
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Strategy: Clone gene next to the promoter of a gene specifically expressed in mammary cells
Vector injected into oocyte, fertilized and implanted
Protein is purified out of milk
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Transgenic plants
Somewhat easier because plant cells are totipotent
Cloned genes can be introduced into somatic tissue and entire plant regenerated using hormones
Agrobacterium tumefaciens naturally infects plant cells and causes tumors Contains Ti plasmid that integrates into host chromosome Codes for plant growth hormones that form crown gall tumor
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Transgenic plants
Ti plasmid modified to introduce cloned genes KanR used as a selectable marker for kanamycin resistance Contain convenient restriction sites
Transformed cells plated on media with kanamycin (to kill nontransformed cells) and carbenicillin(to kill Agrobacterium)
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Successful example of the use of transgenic plants has involved the introduction of genes from Bacillus thuringiensis (Bt)
Bacterium produces toxins that kill certain caterpillars and beetles and has been widely used as an insecticide for several decades
Bt varieties of plants produce the toxins themselves – therefore are resistant to many caterpillars and beetles
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BIOLOGY PRINCIPLE
Biology affects our society
The field of agriculture has been greatly influenced by the development of genetically modified organisms (GMOs) such as Bt corn.
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Cloning in mammals
Identical twins are genetic clones from a single fertilized egg that split early in development
Plants can be cloned from somatic cells
For decades it was believed that mammalian somatic cells could not be used for cloning
1996, Dolly was the first cloned lamb
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Mammary cells from adult sheep removed
Fused diploid mammary cell with enucleated sheep oocyte
Zygote implanted
Dolly and donor were almost genetically identical Same set of genes Minor differences due to differences in
mitochondrial DNA and maternal effect genes
Cloning achieved now in several mammal species
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Stem cells
Have potential to treat a variety of human disorders
Stem cells are undifferentiated cells that can divide and create the bodies of plants and animals They have the capacity to divide Can differentiate into specialized cell types
Embryonic stem cells (ES cells) and embryonic germ cells (EG cells) are pluripotent They can differentiate into many different cell types Thus potential for many different diseases
Stem cells already in use in bone marrow transplants51
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Blaese and colleagues performed the first gene therapy to treat ADA deficiency
Gene therapy puts cloned genes into living cells to cure disease
Over 4,000 diseases involve mutations in single genes
Adenosine deaminase deficiency prevents proper metabolism of nucleosides
Deoxyadenosine accumulates and results in destruction of B and T cells Leads to severe combined immunodeficiency syndrome (SCID) Fatal at an early age (1 or 2 years old)
FEATURE INVESTIGATION
Three treatment approaches: Bone marrow transplant Purified ADA enzyme Gene therapy
Gene therapy September 14, 1990 Removed lymphocytes from girl Treated with retroviral vector containing ADA gene Returned cells to her bloodstream
Results suggest that this first gene therapy trial may offer benefit, but patients were also treated with ADA – so could not know for sure
FEATURE INVESTIGATION
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Retrovirus
HYPOTHESIS Infecting lymphocytes with a retrovirus containing the normal ADA gene will correct the inherited deficiency of the mutant ADA gene in patients with ADA deficiency.
KEY MATERIALS A retrovirus with the normal ADA gene.
Remove ADA-deficient lymphocytes from thepatient with severe combined immunodeficiencydisease (SCID).
Culture the cells in a laboratory.
Lymphocyte
Infuse the ADA-gene-corrected lymphocytes backinto the SCID patient.
LymphocytesLymphocyte
X
Mutant ADA gene
Normal ADA gene
Retroviral inserted DNA
X
Experimental level Conceptual level
3 Infect the lymphocytes with a retrovirus that contains the normal ADA gene. As described in Chapter 18, retroviruses insert their DNA into the host cell chromosome as part of their reproductive cycle.
Retrovirus withADA gene
FEATURE INVESTIGATION
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AD
A f
un
ctio
n f
rom
gen
e th
erap
y(n
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les
of
dea
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ated
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0 365 730 1,095 1,460
Protocol day
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5
0
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5 CONCLUSION The introduction of a cloned ADA gene into lymphocytes via gene therapy resulted in higher ADA function, even after 4 years.
SOURCE Blaese, R. Michael et al. 1995. T lymphocyte- directed gene therapy for ADA-SCID: Initial trial results after 4 years. Science 270:475–480.
THE DATA
Transfusions stopped
FEATURE INVESTIGATION