DNA Technology and Genomics Wilson Muse Schoolcraft College

DNA Technology and Genomics

Wilson MuseSchoolcraft College

GENE CLONING

Copyright © 2009 Pearson Education, Inc.

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§ Plasmid DNA is isolated

§ DNA containing the gene of interest is isolated

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

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

§ Plasmid and target DNA are mixed and associate with each other


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

§ The recombinant DNA is taken up by a bacterial cell

§ The bacterial cell reproduces to form a clone of cells


Animation: Cloning a Gene

Examples ofgene use

RecombinantDNAplasmid

E. coli bacteriumPlasmid

Bacterialchromosome

Gene of interestDNA

Geneof interest

Cell with DNAcontaining geneof interest

Recombinantbacterium

Cloneof cells

Genes may be insertedinto other organisms

Genes or proteinsare isolated from thecloned bacterium

Harvestedproteinsmay be used directly

Examples ofprotein use

Gene of interest

Isolateplasmid

1

IsolateDNA

2

Cut plasmidwith enzyme

3

Cut cell’s DNAwith same enzyme

4

Combine targeted fragmentand plasmid DNA

5

Add DNA ligase,which closesthe circle withcovalent bonds

6

Put plasmidinto bacteriumby transformation

7

Allow bacteriumto reproduce

8

9


Bacterialchromosome

Gene of interestDNA


Isolateplasmid

IsolateDNA

1

2


Bacterialchromosome

Gene of interestDNA


Gene of interest

Isolateplasmid

IsolateDNA



1

2

3

4


Bacterialchromosome

Gene of interestDNA


Gene of interest

Isolateplasmid

IsolateDNA



1

2

3

4


5


Bacterialchromosome

Gene of interestDNA


Gene of interest

Isolateplasmid

IsolateDNA



1

2

3

4


Geneof interest


Add DNA ligase,which closesthe circle withcovalent bonds

5

6


Geneof interest



7


Geneof interest


Cloneof cells



8

7


Geneof interest


Cloneof cells

Genes or proteinsare isolated from thecloned bacterium

Harvestedproteinsmay be used directly

Examples ofprotein use



8

7

Genes may be insertedinto other organisms

Examples ofgene 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 togetherAnimation: Restriction Enzymes

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 molecule5

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 mRNAand addition of reversetranscriptase; synthesisof DNA strand

RNA splicing2

Transcription1

3

Breakdown of RNA4

Synthesis of secondDNA strand

5

mRNA

DNA ofeukaryoticgene

IntronExon

RNAtranscript

Exon Intron Exon

Reverse transcriptase

Test tube

cDNA strandbeing 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

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

GMsheep

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

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

Tiplasmid Insertion of gene

into plasmid

RecombinantTi plasmid

1

Restriction site




into plasmid


1

Restriction site

Plant cell

Introductioninto plantcells

2

DNA carrying new gene




into 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

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

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 questions

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 similar to DNA Fingerprinting

Can be used to assign paternity or link DNA evidence to a crime scene

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 sceneDNA isolated1

Suspect 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

see animation: http://www.dnalc.org/ddnalc/resources/pcr.html

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

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

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

Restrictionenzymes added

DNA sample 1 DNA sample 2

Cut

w

z

x

yy

CutCut

w

z

x

yy

Longerfragments

Shorterfragments

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

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%)

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

Applied by J. Craig Venter and Celera vs traditional method

Venter’s method

– 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. Given alternative splicing and post-translational modifications, the diversity is quite large.

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

How different are we?

Wrap up and Review

• PCR

• Recombinant DNA and Plasmid cloning

• DNA Fingerprinting and Profiling

• DNA sequenceing

Bacterialclone

DNAfragments

Cut Bacterium

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

Bacterialplasmids

(a)

(b)

are copied via

treated with

DNAamplified

via

treated with

DNAsample

(c)

DNAfragments

sorted by size via

Recombinant plasmidsare insertedinto bacteria

Add

(d)

ParticularDNA

sequencehighlighted

(e)

Collectionis called a

(f)

Bacterialplasmids

(a)

(b)

treated with

DNAamplified

via

treated with

DNAsample

are copied via

(c)

DNAfragments

sorted by size via

Recombinant plasmidsare insertedinto bacteria

Add

(d)

ParticularDNA

sequencehighlighted

(e)

Collectionis called a

(f)

(b)

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

§ 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

§ Describe how genes are cloned

You should now be able to

§ Describe how a probe is used to identify a gene of interest

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

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

§ Identify advantages to producing pharmaceuticals with recombinant DNA technology


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

§ Explain how PCR provides copies of a specific DNA sequence

§ Identify ethical concerns related to the use of recombinant DNA technology

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


Documents

DNA Technology and Genomics Wilson Muse Schoolcraft College