Chapter 12

DNA Technology and Genomics

PowerPoint Lectures for Biology: Concepts and Connections, Fifth Edition Campbell, Reece, Taylor, and Simon

Lectures by Chris Romero

Copyright 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

Intro to Genetic Engineering

10.22 Bacteria can transfer DNA in three ways

Bacteria can transfer genes from cell to cell by one of three processes Transformation, transduction, or conjugation
Mating bridge DNA enters cell Phage Phage

Fragment of DNA from another bacterial cell

Bacterial chromosome (DNA)

Fragment of DNA from another bacterial cell (former phage host)

Sex pili

Donor cell (male)

Recipient cell (female)

Figure 10.22AC
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 Once new DNA gets into a bacterial cell Part of it may then integrate into the recipients chromosome
Donated DNA
Crossovers Degraded DNA

Figure 10.22D

Recipient cells chromosome

Recombinant chromosome

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10.23 Bacterial plasmids can serve as carriers for gene transfer

Plasmids Are small circular DNA molecules separate from the bacterial chromosome

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 Plasmids can serve as carriers For the transfer of genes

F factor (plasmid) F factor (integrated)

Male (donor) cell Origin of F replication Bacterial chromosome F factor starts replication and transfer of chromosome
Recipient cell

Male (donor) cell Bacterial chromosome

F factor starts replication and transfer

Only part of the chromosome transfers

Plasmid completes transfer and circularizes Plasmids

Recombination can occur Cell now male Figure 10.23AC

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Colorized TEM 2,000

BACTERIAL PLASMIDS AND GENE CLONING 12.1 Plasmids are used to customize bacteria: An overview
Gene cloning is one application of DNA technology Methods for studying and manipulating genetic material

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 Researchers can insert desired genes into plasmids, creating recombinant DNA And insert those plasmids into bacteria
Bacterium
1 Plasmid

isolated
2 DNA

Cell containing gene of interest isolated

3 Gene inserted

into plasmid Plasmid Bacterial chromosome Recombinant DNA (plasmid) DNA Gene of 4 Plasmid put into interest bacterial cell

Recombinant bacterium
5 Cell multiplies with

gene of interest Copies of gene Copies of protein

Gene for pest resistance inserted into plants

Clone of cells

Protein used to make snow form at higher temperature

Figure 12.1

Gene used to alter bacteria for cleaning up toxic waste

Protein used to dissolve blood clots in heart attack therapy

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 If the recombinant bacteria multiply into a clone The foreign genes are also copied

Can insert regulatory sequences to turn on foreign gene expression in the clone
Can isolate & purify the gene product

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12.3 Genes can be cloned in recombinant plasmids: A closer look

Bacteria take the recombinant plasmids from their surroundings And reproduce, thereby cloning the plasmids and the genes they carry

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 Cloning a gene in a bacterial plasmid

E.coli Isolate DNA 1 from two sources Cut both DNAs 2 with the same restriction enzyme Plasmid Gene V Sticky ends 3 Mix the DNAs; they join by base-pairing DNA Human cell

Add DNA ligase to bond the DNA covalently

Recombinant DNA plasmid

Gene V

Put plasmid into bacterium by transformation

Recombinant bacterium 6 Clone the bacterium

Figure 12.3

Bacterial clone carrying many copies of the human gene

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CONNECTION
12.6 Recombinant cells and organisms can massproduce gene products
Applications of gene cloning include The mass production of gene products for medical and other uses

Table 12.6
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 Different organisms, including bacteria, yeast, and mammals Can be used for this purpose

Figure 12.6
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CONNECTION 12.7 DNA technology is changing the pharmaceutical industry

DNA technology Is widely used to produce medicines and to diagnose diseases

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Therapeutic hormones
In 1982, humulin, human insulin produced by bacteria Became the first recombinant drug approved by the Food and Drug Administration

Figure 12.7A
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Diagnosis and Treatment of Disease

DNA technology

Is being used increasingly in disease diagnosis

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Vaccines
DNA technology

Is also helping medical researchers develop vaccines

Figure 12.7B
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RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 12.8 Nucleic acid probes identify clones carrying specific genes
DNA technology methods Can be used to identify specific pieces of DNA

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 A nucleic acid probe Is a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA Can tag a desired gene in a library
Radioactive probe (DNA) Mix with singlestranded DNA from various bacterial (or phage) clones Single-stranded DNA

Figure 12.8
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Base pairing indicates the gene of interest

CONNECTION 12.9 DNA microarrays test for the expression of many genes at once
DNA microarray assays Can reveal patterns of gene expression in different kinds of cells

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 DNA microarray
DNA microarray Actual size (6,400 genes)

Each well contains DNA from a particular gene

1 mRNA isolated Reverse transcriptase and fluorescent DNA nucleotides 2 cDNA made from mRNA

4 Unbound cDNA rinsed away 3 cDNA applied to wells cDNA Fluorescent spot Nonfluorescent spot

DNA of an expressed gene

Figure 12.9

DNA of an unexpressed gene

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12.10 Gel electrophoresis sorts DNA molecules by size

Mixture of DNA molecules of different sizes

Longer molecules

Power source Gel Shorter molecules

+
Figure 12.10

Completed gel

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12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

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How Restriction Fragments Reflect DNA Sequence

Restriction fragment length polymorphisms (RFLPs)

Reflect differences in the sequences of DNA samples

Crime scene Suspect

w Cut C C G G G G C C z x A C G G T G C C

Cut y

C C G G

G G C C

Cut y

C C G G

G G C C

Figure 12.11A
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DNA from chromosomes

 After digestion by restriction enzymes The fragments are run through a gel

Longer fragments z 1 2

w Shorter fragments
Figure 12.11B

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Using DNA Probes to Detect Harmful Alleles

Radioactive probes

Can reveal DNA bands of interest on a gel

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 Detecting a harmful allele using restriction fragment analysis

1 Restriction fragment

preparation I Restriction fragments

2 Gel electrophoresis

II

III

I II III

3 Blotting

Filter paper

4 Radioactive probe

Radioactive, singlestranded DNA (probe) Probe

5 Detection of radioactivity

(autoradiography)

I II III

Film I II III

Figure 12.11C
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CONNECTION 12.12 DNA technology is used in courts of law

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 DNA fingerprinting can help solve crimes

Defendants blood

Blood from defendants clothes

Victims blood

Figure 12.12A

Figure 12.12B

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DNA and Crime Scene Investigations

Many violent crimes go unsolved

For lack of enough evidence

If biological fluids are left at a crime scene

DNA can be isolated from them

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 DNA fingerprinting is a set of laboratory procedures That determines with near certainty whether two samples of DNA are from the same individual That has provided a powerful tool for crime scene investigators

Investigator at one of the crime scenes (above), Narborough, England (left)

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12.14 The PCR method is used to amplify DNA sequences

The polymerase chain reaction (PCR) Can be used to clone a small sample of DNA quickly, producing enough copies for analysis

Initial DNA segment

Figure 12.14
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Number of DNA molecules

CONNECTION
12.13 Gene therapy may someday help treat a variety of diseases
Gene therapy Is the alteration of an afflicted individuals genes
Cloned gene (normal allele) 1 Insert normal gene into virus Viral nucleic acid Retrovirus 2 Infect bone marrow cell with virus

3 Viral DNA inserts into chromosome Bone marrow cell from patient
Bone marrow

Figure 12.13

4 Inject cells into patient

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 Gene therapy May one day be used to treat both genetic diseases and nongenetic disorders Unfortunately, progress is slow

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GENOMICS CONNECTION
12.15 The Human Genome Project is an ambitious application of DNA technology
The Human Genome Project, begun in 1990 and now largely completed, involved Genetic and physical mapping of chromosomes, followed by DNA sequencing

Figure 12.15
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 The data are providing insight into Development, evolution, and many diseases

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CONNECTION 12.17 The science of genomics compares whole genomes

The sequencing of many prokaryotic and eukaryotic genomes Has produced data for genomics, the study of whole genomes

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 Besides being interesting themselves Nonhuman genomes can be compared with the human genome

Table 12.17

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 Proteomics Is the study of the full sets of proteins produced by organisms

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GENETICALLY MODIFIED ORGANISMS CONNECTION 12.18 Genetically modified organisms are transforming agriculture

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 Recombinant DNA technology Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms
Agrobacterium tumefaciens DNA containing gene for desired trait Plant cell

Ti plasmid

1
Insertion of gene into plasmid using restriction enzyme and DNA ligase

Recombinant Ti plasmid

T DNA Restriction site

Introduction Regeneration into plant of plant cells in culture T DNA carrying new Plant with new trait gene within plant chromosome

Figure 12.18A

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 Transgenic organisms Are those that have had genes from other organisms inserted into their genomes

Figure 12.18B

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 A number of important crops and plants Are genetically modified

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CONNECTION 12.19 Could GM organisms harm human health or the environment?

Development of GM organisms Requires significant safety measures

Figure 12.19A
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 Genetic engineering involves risks Such as ecological damage from GM crops

Figure 12.19B
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CONNECTION 12.20 Genomics researcher Eric Lander discusses the Human Genome Project
Genomics pioneer Eric Lander Points out that much remains to be learned from the Human Genome Project

Figure 12.20
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12.17 The science of genomics compares whole genomes

The sequencing of many prokaryotic and eukaryotic genomes Has produced data for genomics, the study of whole genomes

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The following covers material we did not review during class, but is covered in the textbook. You are not responsible for knowing the following for tests, but it may be useful for understanding the topic.

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12.4 Cloned genes can be stored in genomic libraries

Genomic libraries, sets of DNA fragments containing all of an organisms genes Can be constructed and stored in cloned bacterial plasmids or phages
Recombinant plasmid Genome cut up with restriction enzyme Recombinant phage DNA or

Bacterial clone

Phage clone Phage library

Figure 12.4

Plasmid library

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12.2 Enzymes are used to cut and paste DNA

The tools used to make recombinant DNA are

Restriction enzymes, which cut DNA at specific sequences

DNA ligase, which pastes DNA fragments together

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 Creating recombinant DNA using restriction enzymes and DNA ligase

Restriction enzyme recognition sequence
1

DNA

GAATTC CTTAAG

Restriction enzyme cuts the DNA into fragments

Sticky end Addition of a DNA fragment from another source Two (or more) fragments stick together by base-pairing
4
G A AT T C C T TA A G G A AT T C C T TA A G

DNA ligase pastes the strand

Figure 12.2

Recombinant DNA molecule

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12.5 Reverse transcriptase helps make genes for cloning

Reverse transcriptase can be used to make smaller, complementary DNA (cDNA) libraries Containing only the genes that are transcribed by a particular type of cell
Cell nucleus Exon Intron DNA of eukaryotic gene RNA transcript
2 RNA splicing (removes introns)

Exon

Intron Exon

1 Transcription

mRNA

Test tube Reverse transcriptase cDNA strand

3 Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand

Breakdown of RNA

5 Synthesis of second DNA strand

Figure 12.5

cDNA of gene (no introns)

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12.16 Most of the human genome does not consist of genes

The haploid human genome contains about 25,000 genes And a huge amount of noncoding DNA

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 Much of the noncoding DNA consists of repetitive nucleotide sequences And transposons that can move about within the genome

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