Chapters 20: DNA Technology
AP Biology 2013
Understanding DNAn One of the greatest achievements:
Human Genome Project (completed 2003)
n Sequencing of the genomes of more than 7,000 species was under way in 2010
n Recombinant DNA technology makes this possible (DNA from two species are combined in vitro)
n Genetic Engineering - direct manipulation of genes for practical purposes
n Biotechnology - manipulation of genetic components to make useful products
n Microarray - measures gene expression of many genes at once
Fig. 20.1
DNA Cloningn To work with specific
genes, scientists must identical copies of genes
n Uses plasmids (small circular DNA molecules that replicate separately from the bacterial chromosome)
n Called a cloning vector
n Gene cloning involves using bacteria to make multiple copies of a gene.
Bacterium
Bacterial chromosome
Plasmid
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Gene inserted into plasmid Cell containing gene
of interest
Recombinant DNA (plasmid)
Gene of interest
Plasmid put into bacterial cell
DNA of chromosome (“foreign” DNA)
Recombinant bacterium
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Gene of interest
Protein expressed from gene of interest
Protein harvested Copies of gene
Basic research and various applications
Basic research on protein
Basic research on gene
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hormone treats stunted growth
n Foreign DNA is inserted into a plasmid and the recombinant plasmid is inserted into a bacterial chromosome
Fig. 20.2
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Restriction Enzymesn Restriction enzymes cut
DNA molecules at specific sequences called restriction sites to make restriction fragments
n Most useful restriction enzymes cut in a staggered way producing “sticky ends” that can bond with complementary fragments
n DNA ligase seals the bonds between restriction fragments
Recombinant DNA molecule
One possible combination DNA ligase seals strands
DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.
Restriction enzyme cuts sugar-phosphate backbones.
Restriction site
DNA 5!
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Sticky end
GAATTC CTTAAG
CTTAA G AATTC G
G G AATTC
CTTAA
G G
G G
AATT C AATT C C TTAA C TTAA
Fig. 20.3
Producing Clonesn In gene cloning, the original plasmid is called a cloning vector which is a
DNA molecule that can carry foreign DNA into a host cell and replicate there.
n Ex. hummingbird β-globin gene
n Hummingbird genomic DNA and bacterial plasmid are isolated and cut with the same restriction enzyme
n Fragments are mixed and DNA ligase is added to bond the sticky ends
n Some recombinant plasmids now contain hummingbird DNA
n DNA mixture is added to bacteria
n Bacteria are plated on a type of agar that selects for bacteria with recombinant plasmids
n Results in cloning of many hummingbird DNA fragments (including the β-globin gene)
Producing Clones
Bacterial plasmid TECHNIQUE
RESULTS
ampR gene lacZ gene
Restriction site
Hummingbird cell
Sticky ends Gene of
interest
Humming- bird DNA fragments
Recombinant plasmids Nonrecombinant plasmid
Bacteria carrying plasmids
Colony carrying non- recombinant plasmid with intact lacZ gene
Colony carrying recombinant plasmid with disrupted lacZ gene
One of many bacterial clones Fig. 20.4
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Genomic Librariesn A genomic library is a
collection of recombinant vector clones produced by cloning DNA fragments from an entire genome
n A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a cell (does not include all DNA from the cell)
DNA in nucleus
mRNAs in cytoplasm
mRNA
Reverse transcriptase Poly-A tail
DNA strand
Primer
DNA polymerase
cDNA
5! 5!
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3! 3!
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3! 3!
3! 3!
A A A A A A
A A A A A A
T T T T T
T T T T T
Fig. 20.6
Screening a Genomic Libraryn Clone carrying the gene can be identified with a nucleic acid
probe if its sequence is complimentary to the gene (nucleic acid hybridization)
n Ex. if the desired gene is
n Then the probe would be
5! 3! ⋅⋅⋅ CTCAT CACCGGC⋅⋅⋅
5! 3! G A G T A G T G G C C G
Radioactively labeled probe molecules Gene of
interest Probe DNA
Single- stranded DNA from cell
Film
Location of DNA with the complementary sequence
Nylon membrane
Nylon membrane
Multiwell plates holding library clones
TECHNIQUE 5!
5! 3!
3!
GAGTAGTGGCCG ⋅⋅⋅ CTCATCACCGGC⋅⋅⋅
n DNA probe can be used to screen a large number of clones simultaneously and once identified, the clone carrying the gene can be cultured Fig. 20.7
DNA Amplification
n Polymerase Chain Reaction (PCR)
n Produces many copies of a target segment of DNA
n Uses primers that bracket the desired sequence
Genomic DNA
Target sequence
Denaturation
Annealing
Extension
Primers
New nucleotides
Cycle 1 yields
2 molecules
Cycle 2 yields
4 molecules
Cycle 3 yields 8
molecules; 2 molecules
(in white boxes) match target
sequence
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TECHNIQUE Fig. 20.8
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Restriction Fragment Analysis
n Provides comparative information about DNA sequences
n Useful for comparing two different DNA molecules such as two alleles for a gene.
n Gel electrophoresis - separates nucleic acids or proteins of different lengths or charges
Mixture of DNA mol- ecules of different sizes
Power source
Power source
Longer molecules
Cathode Anode
Wells
Gel
Shorter molecules
TECHNIQUE
RESULTS
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- +
Fig. 20.9
Gel Electrophoresisn Restriction
fragment analysis can be used to compare two different DNA molecules (such as two alleles for a gene)
n Southern blotting combines gel electrophoresis with nucleic acid hybridization
Normal β-globin allele
Sickle-cell mutant β-globin allele
Large fragment
Normal allele
Sickle-cell allele
201 bp 175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of the β-globin gene
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp 175 bp
376 bp
Large fragment
Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
DNA + restriction enzyme
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TECHNIQUE
I Normal β-globin allele
II Sickle-cell allele
III Heterozygote
Restriction fragments Nitrocellulose
membrane (blot)
Heavy weight
Gel
Sponge
Alkaline solution Paper
towels
II I III
II I III II I III
Preparation of restriction fragments
Gel electrophoresis DNA transfer (blotting)
Radioactively labeled probe for β-globin gene
Nitrocellulose blot
Probe base-pairs with fragments
Fragment from sickle-cell β-globin allele
Fragment from normal β- globin allele
Film over blot
Hybridization with labeled probe Probe detection 5 Figs. 20.10 & 20.11
Studying Gene Expressionn Northern Blotting -
combines gel electrophoresis of mRNA by hybridization
n Reverse transcriptase-polymerase chain reaction (RT-PCR) requires less mRNA than Northern blotting
n In situ hydridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in the intact organism
cDNA synthesis
PCR amplification
Gel electrophoresis
mRNAs
cDNAs
Primers
β-globin gene
Embryonic stages 1 2 3 4 5 6
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RESULTS
TECHNIQUE
50 µm
Figs. 20.13
& 20.14
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DNA Microarray
n Allows scientists to automate the measure of expressed genes
n Allows for comparison of gene expression in different tissues, at different times, or under different conditions
Isolate mRNA.
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TECHNIQUE
Make cDNA by reverse transcription, using fluorescently labeled nucleotides.
Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.
Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules (single strands)
DNA fragments representing a specific gene
DNA microarray
DNA microarray with 2,400 human genes
Fig. 20.15
Determining Gene Functionn In vitro mutagenesis
introduces mutations into a cloned gene to disable the function and observe the consequences
n When the mutated gene is returned to the cell the phenotypes can be examined
n Hopefully these techniques can be used to produce stem cells
n Totipotent cell is one that can generate a complete new organism
Frog embryo Frog egg cell Frog tadpole
UV
Less differ- entiated cell
Donor nucleus trans- planted
Enucleated egg cell
Fully differ- entiated (intestinal) cell
Donor nucleus trans- planted Egg with donor nucleus
activated to begin development
Most develop into tadpoles.
Most stop developing before tadpole stage.
EXPERIMENT
RESULTS
Fig. 20.18
Cloning Organismsn Organismal cloning produces
one or more organisms genetically identical to the “parent” that donated the single cell
n Nuclear transplantation - nucleus of an unfertilized egg or zygote is replaced with the nucleus of a differentiated cell
n 1997 - Dolly the sheep was born in Scotland (her death in 2003 and health problems may show that cloned cells are not as healthy)
Mammary cell donor
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TECHNIQUE
RESULTS
Cultured mammary cells
Egg cell from ovary
Egg cell donor
Nucleus removed Cells fused
Grown in culture
Implanted in uterus of a third sheep
Embryonic development
Nucleus from mammary cell
Early embryo
Surrogate mother
Lamb (“Dolly”) genetically identical to mammary cell donor
Fig. 20.19
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Problems with Animal Cloning
n Only a small percentage of cloned embryos have developed normally to birth (many exhibit defects)
n Epigenetic changes (acetylation of histones or methylation of DNA) must be reversed in donor nucleus in order for genes to be expressed appropriately
Stem Cellsn Stem cell is a unspecialized cell
that can reproduce itself indefinitely and differentiate into specialized cells of one or more types
n Stem cells isolated from embryos at the blastocyst stage are called embryonic stem (ES) cells and can differentiate into all cell types
n Adult stem cells can replace non-reproducing specialized cells
n Researchers have used skin cells to produce ES cells by using viruses to introduce stem cell master regulatory genes. These cells are called iPS cells (induced pluripotent stem cells).
Cultured stem cells
Different culture conditions
Different types of differentiated cells
Embryonic stem cells
Adult stem cells
Cells generating all embryonic cell types
Cells generating some cell types
Liver cells
Nerve cells
Blood cells
Remove skin cells from patient. 2
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Reprogram skin cells so the cells become induced pluripotent stem (iPS) cells.
Patient with damaged heart tissue or other disease
Return cells to patient, where they can repair damaged tissue.
Treat iPS cells so that they differentiate into a specific cell type.
Figs. 20.21 & 20.22
Practical Applications
n Identification of genes whose mutation causes genetic diseases
n Diagnosis of genetic disorders
n Gene therapy - alteration of afflicted individual’s genome (uses vectors to deliver genes to cells)
n Productions of pharmaceutical products (ex. hormones, vaccines) using transgenic animals
n DNA fingerprinting (CSI)
n Environmental cleanup
n Agriculture
Cloned gene
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Retrovirus capsid
Bone marrow cell from patient
Viral RNA
Bone marrow
Insert RNA version of normal allele into retrovirus.
Let retrovirus infect bone marrow cells that have been removed from the patient and cultured.
Viral DNA carrying the normal allele inserts into chromosome.
Inject engineered cells into patient.
Fig. 20.23
Fig. 20.24
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