Chapter 12 DNA Technology and Genomics...Chapter 12 DNA Technology and Genomics Lecture by Mary C. Colavito DNA evidence was used to solve a double murder in England –Showed that

Copyright © 2009 Pearson Education, Inc.

PowerPoint Lectures for

Biology: Concepts & Connections, Sixth EditionCampbell, Reece, Taylor, Simon, and Dickey

Chapter 12 DNA Technology and Genomics

Lecture by Mary C. Colavito

DNA evidence was used to solve a double murder in England

– Showed that two murders could have been committed by the same person

– Showed the innocence of someone who confessed to one of the murders

– Showed the absence of a match in 5,000 men tested when the murderer persuaded another man to donate blood in his name

– Showed a match with the murderer and DNA found with both victims

Introduction: DNA and Crime Scene Investigations


GENE CLONING


12.1 Genes can be cloned in recombinant plasmids

Genetic engineering involves manipulating genes for practical purposes

– Gene cloning leads to the production of multiple identical copies of a gene-carrying piece of DNA

– Recombinant DNA is formed by joining DNA sequences from two different sources

– One source contains the gene that will be cloned

– Another source is a gene carrier, called a vector

– Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors


Steps in cloning a gene

1. Plasmid DNA is isolated

2. DNA containing the gene of interest is isolated

3. Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle

4. DNA with the target gene is treated with the same enzyme and many fragments are produced

5. Plasmid and target DNA are mixed and associate with each other



6. Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together

7. The recombinant DNA is taken up by a bacterial cell

8. The bacterial cell reproduces to form a clone of cells



Animation: Cloning a Gene

12_01CloningAGene_A.html12_01CloningAGene_A.html

Examples of

gene use

Recombinant

DNA

plasmid

E. coli bacteriumPlasmid

Bacterial

chromosome

Gene of interest

DNA

Gene

of interest

Cell with DNA

containing gene

of interest

Recombinant

bacterium

Clone

of cells

Genes may be inserted

into other organisms

Genes or proteins

are isolated from the

cloned bacterium

Harvested

proteins

may be

used directly

Examples of

protein use

Gene

of interest

Isolate

plasmid

1

Isolate

DNA

2

Cut plasmid

with enzyme

3

Cut cell’s DNA

with same enzyme

4

Combine targeted fragment

and plasmid DNA

5

Add DNA ligase,

which closes

the circle with

covalent bonds

6

Put plasmid

into bacterium

by transformation

7

Allow bacterium

to reproduce

8

9


Bacterial

chromosome

Gene of interest

DNA

Cell with DNA

containing gene

of interestIsolate

plasmidIsolate

DNA

1

2


Bacterial

chromosome

Gene of interest

DNA

Cell with DNA

containing gene

of interest

Gene

of interest

Isolate

plasmidIsolate

DNA

Cut plasmid

with enzyme

Cut cell’s DNA

with same enzyme

1

2

3

4


Bacterial

chromosome

Gene of interest

DNA

Cell with DNA

containing gene

of interest

Gene

of interest

Isolate

plasmidIsolate

DNA

Cut plasmid

with enzyme

Cut cell’s DNA

with same enzyme

1

2

3

4


and plasmid DNA

5


Bacterial

chromosome

Gene of interest

DNA

Cell with DNA

containing gene

of interest

Gene

of interest

Isolate

plasmidIsolate

DNA

Cut plasmid

with enzyme

Cut cell’s DNA

with same enzyme

1

2

3

4

Recombinant

DNA

plasmidGene

of interest


and plasmid DNA

Add DNA ligase,

which closes

the circle with

covalent bonds

5

6

Recombinant

DNA

plasmidGene

of interest

Recombinant

bacterium

Put plasmid

into bacterium

by transformation

7

Recombinant

DNA

plasmidGene

of interest

Recombinant

bacterium

Clone

of cells

Put plasmid

into bacterium

by transformation

Allow bacterium

to reproduce

8

7

Recombinant

DNA

plasmidGene

of interest

Recombinant

bacterium

Clone

of cells

Genes or proteins

are isolated from the

cloned bacterium

Harvested

proteins

may be

used directly

Examples of

protein use

Put plasmid

into bacterium

by transformation

Allow bacterium

to reproduce

8

7

Genes may be inserted

into other organisms

Examples of

gene use

9

12.2 Enzymes are used to “cut and paste” DNA

Restriction enzymes cut DNA at specific sequences

– Each enzyme binds to DNA at a different restriction site

– Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends”

– Fragments with complementary sticky ends can associate with each other, forming recombinant DNA

DNA ligase joins DNA fragments together


Animation: Restriction Enzymes

12_02RestrictionEnzymes_A.html12_02RestrictionEnzymes_A.html

Restriction enzymerecognition sequence

1

2

DNA

Restriction enzymecuts the DNA intofragments

Sticky end


1

2

DNA


Sticky end

3

Addition of a DNAfragment fromanother source


1

2

DNA


Sticky end

3


4

Two (or more)fragments sticktogether bybase-pairing


1

2

DNA


Sticky end

3


4

Two (or more)fragments sticktogether bybase-pairing

DNA ligasepastes the strands

RecombinantDNA molecule

5

12.3 Cloned genes can be stored in genomic libraries

A genomic library is a collection of all of the cloned DNA fragments from a target genome

Genomic libraries can be constructed with different types of vectors

– Plasmid library: genomic DNA is carried by plasmids

– Phage library: genomic DNA is incorporated into bacteriophage DNA

– Bacterial artificial chromosome (BAC) library: specialized plasmids can carry large DNA sequences


Recombinantplasmid

Phageclone

Bacterialclone

Phage libraryPlasmid library

or

Recombinantphage DNA

Genome cut up withrestriction enzyme

12.4 Reverse transcriptase can help make genes for cloning

Complementary DNA (cDNA) is used to clone eukaryotic genes

– mRNA from a specific cell type is the template

– Reverse transcriptase produces a DNA strand from mRNA

– DNA polymerase produces the second DNA strand

Advantages of cloning with cDNA

– Study genes responsible for specialized characteristics of a particular cell type

– Obtain gene sequences without introns

– Smaller size is easier to handle

– Allows expression in bacterial hosts


Cell nucleus

Isolation of mRNA

and addition of reverse

transcriptase; synthesis

of DNA strand

RNA splicing2

Transcription1

3

Breakdown of RNA4

Synthesis of second

DNA strand

5

mRNA

DNA of

eukaryotic

gene

IntronExon

RNA

transcript

Exon Intron Exon

Reverse transcriptase

Test tube

cDNA strand

being synthesized

cDNA of gene

(no introns)

12.5 Nucleic acid probes identify clones carrying specific genes

Nucleic acid probes bind to cloned DNA

– Probes can be DNA or RNA sequences complementary to a portion of the gene of interest

– A probe binds to a gene of interest by base pairing

– Probes are labeled with a radioactive isotope or fluorescent tag for detection


12.5 Nucleic acid probes identify clones carrying specific genes

Screening a gene library

– Bacterial clones are transferred to filter paper

– Cells are lysed and DNA is separated into single strands

– A solution containing the probe is added, and binding to the DNA of interest is detected

– The clone carrying the gene of interest is grown for further study


RadioactiveDNA probe

Single-strandedDNA

Base pairingindicates thegene of interest

Mix with single-stranded DNA fromgenomic library

GENETICALLY MODIFIED ORGANISMS


12.6 Recombinant cells and organisms can mass-produce gene products

Cells and organisms containing cloned genes are used to manufacture large quantities of gene products

Capabilities of the host cell are matched to the characteristics of the desired product

– Prokaryotic host: E. coli

– Can produce eukaryotic proteins that do not require post-translational modification

– Has many advantages in gene transfer, cell growth, and quantity of protein production

– Can be engineered to secrete proteins


12.6 Recombinant cells and organisms can mass-produce gene products

Capabilities of the host cell are matched to the characteristics of the desired product

– Eukaryotic hosts

– Yeast: S. cerevisiae

– Can produce and secrete complex eukaryotic proteins

– Mammalian cells in culture

– Can attach sugars to form glycoproteins

– “Pharm” animals

– Will secrete gene product in milk


12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine

Products of DNA technology

– Therapeutic hormones

– Insulin to treat diabetes

– Human growth hormone to treat dwarfism

– Diagnosis and treatment of disease

– Testing for inherited diseases

– Detecting infectious agents such as HIV



Products of DNA technology

– Vaccines

– Stimulate an immune response by injecting

– Protein from the surface of an infectious agent

– A harmless version of the infectious agent

– A harmless version of the smallpox virus containing genes from other infectious agents



Advantages of recombinant DNA products

– Identity to human protein

– Purity

– Quantity


12.8 CONNECTION: Genetically modified organisms are transforming agriculture

Genetically modified (GM) organisms contain one or more genes introduced by artificial means

Transgenic organisms contain at least one gene from another species

GM plants

– Resistance to herbicides

– Resistance to pests

– Improved nutritional profile

GM animals

– Improved qualities

– Production of proteins or therapeutics


Agrobacterium tumefaciens

DNA containinggene for desired trait

Ti

plasmid Insertion of geneinto plasmid

Recombinant

Ti plasmid

1

Restriction site



Ti


Recombinant

Ti plasmid

1

Restriction site

Plant cell

Introductioninto plantcells

2

DNA carrying new gene



Ti


Recombinant

Ti plasmid

1

Restriction site

Plant cell

Introductioninto plantcells

2

DNA carrying new gene

Regenerationof plant

3

Plant with new trait

12.9 Genetically modified organisms raise concerns about human and environmental health

Scientists use safety measures to guard against production and release of new pathogens

Concerns related to GM organisms

– Can introduce allergens into the food supply

– FDA requires evidence of safety before approval

– Exporters must identify GM organisms in food shipments

– May spread genes to closely related organisms

– Hybrids with native plants may be prevented by modifying GM plants

Regulatory agencies address the safe use of biotechnology


12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases

Gene therapy aims to treat a disease by supplying a functional allele

One possible procedure

– Clone the functional allele and insert it in a retroviral vector

– Use the virus to deliver the gene to an affected cell type from the patient, such as a bone marrow cell

– Viral DNA and the functional allele will insert into the patient’s chromosome

– Return the cells to the patient for growth and division


12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases

SCID (severe combined immune deficiency) was the first disease treated by gene therapy

– First trial in 1990 was inconclusive

– Second trial in 2000 led to the development of leukemia in some patients due to the site of gene insertion

Challenges

– Safe delivery to the area of the body affected by the disease

– Achieving a long-lasting therapeutic effect

– Addressing ethical questionsCopyright © 2009 Pearson Education, Inc.

Insert normal geneinto virus

1

Viral nucleic acid

Retrovirus

Infect bone marrowcell with virus

2

Viral DNA insertsinto chromosome

3

Inject cellsinto patient

4

Bone marrowcell from patient

Bonemarrow

Cloned gene(normal allele)

DNA PROFILING


12.11 The analysis of genetic markers can produce a DNA profile

DNA profiling is the analysis of DNA fragments to determine whether they come from a particular individual

– Compares genetic markers from noncoding regions that show variation between individuals

– Involves amplification (copying) of markers for analysis

– Sizes of amplified fragments are compared


Crime scene

DNA isolated1Suspect 1 Suspect 2

DNA of selectedmarkers amplified

2

Amplified DNA compared

3

12.12 The PCR method is used to amplify DNA sequences

Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule

Relies upon a pair of primers– Short DNA molecules that bind to sequences at each

end of the sequence to be copied

– Used as a starting point for DNA replication

Repeated cycle of steps for PCR– Sample is heated to separate DNA strands

– Sample is cooled and primer binds to specific target sequence

– Target sequence is copied with heat-stable DNA polymerase


Advantages of PCR

– Can amplify DNA from a small sample

– Results are obtained rapidly

– Reaction is highly sensitive, copying only the target sequence


12.12 The PCR method is used to amplify DNA sequences

Cycle 1yields 2 molecules

21 3

GenomicDNA



3 5 3 5 3 5

Targetsequence

Heat toseparateDNA strands

Cool to allowprimers to formhydrogen bondswith ends oftarget sequences

35

3 5

35

35 35

Primer New DNA

5

DNApolymerase addsnucleotidesto the 3 endof each primer

5


GenomicDNA

3 5 3 5 3 5

Targetsequence

Heat toseparateDNA strands

Cool to allowprimers to formhydrogen bondswith ends oftarget sequences

35

3 5

35

35 35

Primer New DNA

5

DNApolymerase addsnucleotidesto the 3 endof each primer

215

3

12.13 Gel electrophoresis sorts DNA molecules by size

Gel electrophoresis separates DNA molecules based on size

– DNA sample is placed at one end of a porous gel

– Current is applied and DNA molecules move from the negative electrode toward the positive electrode

– Shorter DNA fragments move through the gel pores more quickly and travel farther through the gel

– DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence

– Each band is a collection of DNA molecules of the same length


Video: Biotechnology Lab

12_13BiotechLab_SV.mpg12_13BiotechLab_SV.mpg

Mixture of DNAfragments ofdifferent sizes

Completed gel

Longer(slower)molecules

Gel

Powersource

Shorter(faster)molecules

12.14 STR analysis is commonly used for DNA profiling

Short tandem repeats (STRs) are genetic markers used in DNA profiling

– STRs are short DNA sequences that are repeated many times in a row at the same location

– The number of repeating units can differ between individuals

– STR analysis compares the lengths of STR sequences at specific regions of the genome

– Current standard for DNA profiling is to analyze 13 different STR sites


STR site 1

Crime scene DNA

STR site 2

Suspect’s DNA

Number of short tandemrepeats match

Number of short tandemrepeats do not match

Crime sceneDNA

Suspect’sDNA

12.15 CONNECTION: DNA profiling has provided evidence in many forensic investigations

Forensics

– Evidence to show guilt or innocence

Establishing family relationships

– Paternity analysis

Identification of human remains

– After tragedies such as the September 11, 2001, attack on the World Trade Center

Species identification

– Evidence for sale of products from endangered species


12.16 RFLPs can be used to detect differences in DNA sequences

Single nucleotide polymorphism (SNP) is a variation at one base pair within a coding or noncoding sequence

Restriction fragment length polymorphism(RFLP) is a variation in the size of DNA fragments due to a SNP that alters a restriction site

– RFLP analysis involves comparison of sizes of restriction fragments by gel electrophoresis


Restriction

enzymes added

DNA sample 1 DNA sample 2

Cut

w

z

x

yy

CutCut

w

z

x

yy

Longerfragments

Shorterfragments

GENOMICS


12.17 Genomics is the scientific study of whole genomes

Genomics is the study of an organism’s complete set of genes and their interactions

– Initial studies focused on prokaryotic genomes

– Many eukaryotic genomes have since been investigated

Evolutionary relationships can be elucidated

– Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans

– Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast


12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes

Goals of the Human Genome Project (HGP)

– To determine the nucleotide sequence all DNA in the human genome

– To identify the location and sequence of every human gene


12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes

Results of the Human Genome Project– Humans have 21,000 genes in 3.2 billion nucleotide

pairs

– Only 1.5% of the DNA codes for proteins, tRNAs, or rRNAs

– The remaining 88.5% of the DNA contains– Control regions such as promoters and enhancers

– Unique noncoding DNA

– Repetitive DNA

– Found in centromeres and telomeres

– Found dispersed throughout the genome, related to transposable elements that can move or be copied from one location to another


RepetitiveDNA thatincludestransposableelementsand relatedsequences(44%)

RepetitiveDNA unrelated totransposableelements(15%)

UniquenoncodingDNA (15%)

Introns andregulatorysequences(24%)

Exons (regions of genes coding for proteinor giving rise to rRNA or tRNA) (1.5%)

12.19 The whole-genome shotgun method of sequencing a genome can provide a wealth of data quickly

Three stages of the Human Genome Project

– A low-resolution linkage map was developed using RFLP analysis of 5,000 genetic markers

– A physical map was constructed from nucleotide distances between the linkage-map markers

– DNA sequences for the mapped fragments were determined


12.19 The whole-genome shotgun method of sequencing a genome can provide a wealth of data quickly

Whole-genome shotgun method

– Restriction enzymes were used to produce fragments that were cloned and sequenced

– Computer analysis assembled the sequence by aligning overlapping regions


Chop up withrestriction enzyme

Chromosome

DNA fragments

Sequencefragments

Alignfragments

Reassemblefull sequence

12.20 Proteomics is the scientific study of the full set of proteins encoded by a genome

Proteomics

– Studies the proteome, the complete set of proteins specified by a genome

– Investigates protein functions and interactions

The human proteome may contain 100,000 proteins


12.21 EVOLUTION CONNECTION: Genomes hold clues to the evolutionary divergence of humans and chimps

Comparisons of human and chimp genomes

– Differ by 1.2% in single-base substitutions

– Differ by 2.7% in insertions and deletions of larger DNA sequences

– Human genome shows greater incidence of duplications

– Genes showing rapid evolution in humans

– Genes for defense against malaria and tuberculosis

– Gene regulating brain size

– FOXP2 gene involved with speech and vocalization


Bacterialclone

DNAfragments

CutBacterium

Recombinantbacteria

Genomic library

RecombinantDNA

plasmids

Cut

Plasmids

Longer fragmentsmove slower

Powersource

Shorter fragmentsmove faster

Mixture of DNA fragments

A “band” is acollection of DNAfragments of oneparticular length

DNA attracted to +pole due to PO4

– groups

Bacterial

plasmids

(a)

(b)

are copied via

treated with

DNA

amplified

via

treated with

DNA

sample

(c)

DNA

fragments

sorted by size via

Recombinant plasmids

are inserted

into bacteria

Add

(d)

Particular

DNA

sequence

highlighted

(e)

Collection

is called a(f)

Bacterial

plasmids

(a)

(b)

treated with

DNAamplified

via

treated with

DNA

sample

are copied via

(c)

DNA

fragments

sorted by size via

Recombinant plasmids

are inserted

into bacteria

Add

(d)

Particular

DNA

sequence

highlighted

(e)

Collection

is called a(f)

(b)

1.Distinguish between terms in the following groups: restriction enzyme—DNA ligase; GM organism—transgenic organism; SNP—RFLP; genomics—proteomics

2.Define the following terms: cDNA, gel electrophoresis, gene cloning, genomic library, “pharm” animal, plasmid, probe, recombinant DNA, repetitive DNA, reverse transcriptase, STR, Taq polymerase, vector, whole-genome shotgun method

3.Describe how genes are cloned

You should now be able to


4.Describe how a probe is used to identify a gene of interest

5.Describe how gene therapy has been attempted and identify challenges to the effectiveness of this treatment approach

6.Distinguish between the use of prokaryotic and eukaryotic cells in producing recombinant DNA products

7.Identify advantages to producing pharmaceuticals with recombinant DNA technology



8. Describe the basis for DNA profiling and explain how it is used to provide evidence in forensic investigations

9. Explain how PCR provides copies of a specific DNA sequence

10. Identify ethical concerns related to the use of recombinant DNA technology

11. Describe how comparative information from genome projects has led to a better understanding of human biology



Documents

Chapter 12 DNA Technology and Genomics...Chapter 12 DNA Technology and Genomics Lecture by Mary C. Colavito DNA evidence was used to solve a double murder in England –Showed that