dna technologies. biotechnology dolly and surrogate mom genetically modified rice. embryonic stem...

DNA Technologies

Biotechnology

Dolly and surrogate Mom

Genetically modified rice.

Embryonic stem cells and gene therapy

Biotechnology

Biotechnology, defined broadly, is the engineering of organisms for useful purposes.

Often, biotechnology involves the creation of hybrid genes and their introduction into organisms in which some or all of the gene is not normally present.

Fourteen month-old genetically engineered (“biotech”) salmon (left) and standard salmon (right).

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DNA technology is the chemical manipulation of the genotypes and resulting phenotypes of organisms such that living organisms are modified

Alternatively, no-longer-living organisms or their no-longer-living parts may be analyzed chemically at the level of genotype

DNA technology has revolutionized how scientists study the genetics, biochemistry, even the ecology and evolutionary biology of organisms

Genetic engineering is the artificial manipulation of the genetic material of organisms, including the creation of novel genetic material (i.e., novel nucleotide sequences)

Biotechnology is the development of novel biological products, indeed whole industries are now devoted to the production and analysis of biological materials

Biotechnology

We’ll examine:

Animal cloning

Gene cloning for pharmaceutical production

The promise and perhaps perils of embryonic stem cells

DNA fingerprinting

Genetically modified foods and the American-European opinion divide.

Animal Cloning

Dolly and her surrogate mother.

Why Clone Animals?

To answer questions of basic biology

Five genetically identical cloned pigs.

For herd improvement. To satisfy our desires (e.g. pet cloning).

For pharmaceutical production.

Is Animal Cloning Ethical?

The first cloned horse and her surrogate mother/genetic twin.

As with many important questions, the answer is beyond the scope of science.

USU’s Contribution – A Cloned Mule and the First Cloned Equine

The Biotechnology of Reproductive Cloning

Even under the best of circumstances, the current technology of cloning is very inefficient.

Cloning provides the most direct demonstration that all cells of an individual share a common genetic blueprint.

Saved by Cloning?

Some are firm believers while many view these approaches to be more of a stunt.

Note the use of a closely related species, a domestic goat, as egg donor and surrogate mother.

(Science (2002) 295:1443)

Carbon Copy– the First Cloned Pet

Significantly, Carbon Copy is not a phenotypic carbon copy of the animal she was cloned from.

The Next Step?

Highly unlikely.

Attempts at human cloning are viewed very unfavorably in the scientific community.

So how do we do all of this???

Recombinant DNA methods Restriction enzymes

Enzymes from bacteria Used to cut DNA molecules in specific places Enable researchers to cut DNA into

manageable segments Vector -molecule carrier of DNA fragment into

cell Transformation: uptake of foreign DNA into cells

Cutting DNA with a restriction enzyme

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Restriction Endonucleases

• A Restriction Endonucleases will cut both strands of a DNA duplex at a specific place

• These “places” need not be directly opposite:

• Note that the above enzyme is EcoRI, the first restriction endonuclease characterized

5’…GAATTC…3’ 3’…CTTAAG…5’

5’…GAATTC…3’ 3’…CTTAAG…5’

5’…G -OH P-AATTC…3’ 3’…CTTAA -P HO-G…5’

5’…G -OH P-AATTC…3’ 3’…CTTAA -P HO-G…5’

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Most R.E. RecognitionSequences are Palindromes

G^AATT-CC-TTAA^G

G^GATC-CC-CTAG^G

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GC^GGCC-GCCG-CCGG^CG

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Enzyme Sequence Product

EcoRI G^AATTC 5’ sticky ends

BamHI G^GATCC 5’ sticky ends

Bg1II A^GATCT 5’ sticky ends

PvuI CGATC^G 3’ sticky ends

PvuII CAG^CTG Blunt end

MboI G^ATC 5’ sticky ends

HindIII A^AGCTT 5’ sticky ends

HinfI G^ANTC 5’ sticky ends

Sau3A G^ATC 5’ sticky ends

AluI AG^CT Blunt end

TaqI T^CGA 5’ sticky ends

HaeIII G^GCC 5’ sticky ends

NofI GC^GGCCGC 5’ sticky ends

Restriction Enzymes are Enzymes That Cut DNA Only at Particular Sequences

The enzyme EcoRI cutting DNA at its recognition sequence

Different restriction enzymes have different recognition sequences.

This makes it possible to create a wide variety of different gene fragments.

Restriction enzyme animation

Recombinant DNA, Gene Cloning, and Pharmaceutical Production

DNA can be cut at specific sequences using restriction enzymes.

This creates DNA fragments useful for gene cloning.

These are mature and widely utilized biotechnologies.

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Upon ligation we now have recombinant

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Splicing foreign DNA into a vector Foreign DNA and plasmid DNA cut with same restriction

enzyme Produces linear molecules with complementary single-

stranded ends Recombinant DNA created by mixing so sticky ends pair DNA ligase forms covalent bonds, linking the two fragments

DNAs Cut by a Restriction Enzyme Can be Joined Together in New Ways

These are recombinant DNAs and they often are made of DNAs from different organisms.

Plasmids

Plasmids are Used to Replicate a Recombinant DNA

Plasmids are small circles of DNA found in bacteria.

Plasmids replicate independently of the bacterial chromosome.

Replication often produces 50-100 copies of a recombinant plasmid in each cell.

Pieces of foreign DNA can be added within a plasmid to create a recombinant plasmid.

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Note that plasmid is vector that

carries DNA into recipient

cells via transformation

Other vectors include viruses (transduction)

as well as otherwise inert

projectiles

Harnessing the Power of Recombinant DNA Technology – Human Insulin Production by Bacteria

http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/plasmidcloning_fla.html

animation

Human Insulin Production by Bacteria

6) join the plasmid and human fragment

and cut with a restriction enzyme

Human Insulin Production by Bacteria

Mix the recombinant plasmid with bacteria.

Screening bacterial cells to learn which contain the human insulin gene is the hard part.

Route to the Production by Bacteria of Human Insulin

A fermentor used to grow recombinant bacteria.

This is the step when gene cloning takes place.

The single recombinant plasmid replicates within a cell.

Then the single cell with many recombinant plasmids produces trillions of like cells with recombinant plasmid – and the human insulin gene.

One cell with the recombinant plasmid

Route to the Production by Bacteria of Human Insulin

The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene.

Route to the Production by Bacteria of Human Insulin

Overview of gene cloning.

Cloning animation

Pharming

These goats contain the human gene for a clot-dissolving protein that is produced in their milk.

Pharming is the production of pharmaceuticals in animals engineered to contain a foreign, drug-producing gene.

Genetically Modified Foods

Many of our crops in the US are genetically modified.

Should they be?

GM Crops are Here Today

Source: Pew Initiative on Food and Biotechnology, August 2004.

Methods for Plant Genetic Engineering are Well-Developed and Similar to Those for Animals

Golden Rice is Modified to be Provide a Dietary Source of Vitamin A

Worldwide, 7% of children suffer vitamin A deficiency, many of them living in regions in which rice is a staple of the diet.

Golden rice (yellow) with standard rice (white).

Genetically Modified Crops

Genetically Modified Cotton (contains a bacterial gene for pest resistance)

Standard Cotton

GMOs, Especially Outside the US, Are a Divisive Issue

Protesters at the 2000 Montreal World Trade Summit

European sentiment

Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops

The jury’s still out on the magnitude of GM crop’s ecological impact, but the question is debated seriously.

Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops

Genetic probes Segments of single-stranded DNA that can hybridize to

complementary base sequences in target gene Usually a radioactive piece that “labels” the DNA Southern blot technique

Using a geneticprobe to find bacterial cellswith a specificrecombinant DNA molecule

Amplifying DNA in vitro by PCR Small amount of double-stranded DNA DNA precursors Specific nucleic acid primers Taq DNA polymerase

DNA is denatured Primers attach to primer-binding site on each DNA strand Each strand acts as template for DNA synthesis

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Cloning allows the amplification

of genotype (DNA) as well as

phenotype (proteins)

If all you really need is the DNA, then PCR is an

easy way to amplify DNA

without cloning

Amplification of DNA by PCR

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Gel Electrophoresis

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DNA, the Law, and Many Other Applications – The Technology of DNA Fingerprinting

A DNA fingerprint used in a murder case.

What are we looking at? How was it produced?

The defendant stated that the blood on his clothing was his.

DNA Fingerprinting Basics

Different individuals carry different alleles.

Most alleles useful for DNA fingerprinting differ on the basis of the number of repetitive DNA sequences they contain.

DNA Fingerprinting Basics

If DNA is cut with a restriction enzyme that recognizes sites on either side of the region that varies, DNA fragments of different sizes will be produced.

A DNA fingerprint is made by analyzing the sizes of DNA fragments produced from a number of different sites in the genome that vary in length.

The more common the length variation at a particular site and the greater the number the sites analyzed, the more informative the fingerprint.

A Site With Three Alleles Useful for DNA Fingerprinting

DNA fragments of different size will be produced by a restriction enzyme that cuts at the points shown by the arrows.

The DNA Fragments Are Separated on the Basis of Size

The technique is gel electrophoresis.

The pattern of DNA bands is compared between each sample loaded on the gel.

Gel electrophoresis animation

Possible Patterns for a Single “Gene” With Three Alleles

In a standard DNA fingerprint, about a dozen sites are analyzed, with each site having many possible alleles.

A DNA Fingerprint

When many genes are analyzed, each with many different alleles, the chance that two patterns match by coincidence is vanishingly small.

DNA detective animation

HGP fingerprinting page

DNA and the Law

SLT 3/8/05

Some applications of DNA fingerprinting in the justice system.

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DNA typing

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Northern Blot

A Northern Blot is the same as a Southern Blot-except instead of probing for DNA- the prob is for RNA

Used to detect whether or not transcription is occuring

Western Blot

Same as Southern/Northern except the probe is for protein

Used to detect whether a particular gene is being “turned on” or “off”

DNA sequencing Based on chain termination method Yields information about

Structure of gene Probable amino acid sequences of its encoded proteins

Chain termination method of DNA sequencing

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Transgenic organisms Foreign DNA incorporated into their genetic material Gene target

Single gene inactivated or “knocked out” “Knockout mice” used in studying genes

Mutagenesis screening Male mice treated with mutagens and studied

A transgenicmouse

Mouse on rightis normal; mouseon left is transgenicanimal expressingrat growth hormone

Transgenicrice

“Golden rice”shown intermixedwith white ricecontain highconcentrationsof beta-carotene

Safety guidelines Safety concerns

Introduction of transgenic organisms into the environment

Health effects on humans from consuming GM crops Safety measures

Special facilities designed to hold pathogenic organisms

Science of risk assessment

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One example of this might

be done… is essentially cloning into

animals

Injecting DNA into Egg

Genome Comparisons

The Stem Cell Concept

A stem cell is an undifferentiated, dividing cell that gives rise to a daughter cell like itself and a daughter cell that becomes a specialized cell type.

Stem Cells are Found in the Adult, but the Most Promising Types of Stem Cells for Therapy are Embryonic Stem Cells

The Inner Cell Mass is the Source of Embryonic Stem Cells

The embryo is destroyed by separating it into individual cells for the collection of ICM cells.

Some Thorny Ethical Questions

Is it ethical to harvest embryonic stem cells from the “extra” embryos created during in vitro fertilization?

Are these masses of cells a human?

The Promise and Possible Perils of Stem Cells

Additional Potential Dilemmas – Therapeutic Cloning to Obtain Matched Embryonic Stem Cells

Cells from any source other than you or an identical twin present the problem of rejection.

If so, how can matched embryonic stem cells be obtained?

A cloned embryo of a person can be made, and embryonic stem cells harvested from these clones.

Cultured mouse embryonic stem cells.

Therapeutic Cloning

Is there any ethical difference between therapeutic and reproductive cloning?

HIV/Pregnancy Tests

Done using body’s own immune response ELISA test

Cells naturally have antigens on their outer surface.

The antibodies respond to the antigens in an immune response

Antibodies and antigens fit together “like a glove”

http://www.elispot-analyzers.de/english/elisa-animation.html

Animation

http://www.genome.gov/Pages/EducationKit/download.htmlDNA technology video

Online Tools

• Buying primers (custom oligos): http://www.qiagen.com/• 2x2 sequence comparisons: http://www2.igh.cnrs.fr/bin/align-

guess.cgi• n x n sequence comparisons:

http://www.genebee.msu.su/services/malign_reduced.html• Sequence manipulation:

http://arbl.cvmbs.colostate.edu/molkit/manip/index.html• Sequence translation:

http://arbl.cvmbs.colostate.edu/molkit/translate/index.html• Restriction Enzymes: http://rebase.neb.com/rebase/rebase.html• Restriction sites:

http://www.ccsi.com/firstmarket/cutter/cut2.html• Sequence searches: http://www.ncbi.nlm.nih.gov/blast/• Other tools: http://molbiol-tools.ca/