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!

5!

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5! 5!

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5! 5!

5!

5!

3!

3!

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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!

5! 5!

5! 5!

5! 5!

3! 3!

3! 3!

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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2

- +

- +

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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