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Biotechnology

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Review of last class What is sequencing?

What is a 23 dideoxynucleotide?

How does the dideoxy method of sequencing work?

2 3 Dideoxynucleotides cause chain termination of

DNA Polymerase

Why did sanger choose X174 ?

Small genome of X174.A

5375bp

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Methods for detecting mRNA

Northern Blotting

RT-PCR

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Northern blotting: gel electrophoresis of mRNAfollowed by hybridization with a probe on a

membrane

Uses: Identification of mRNA at a particular

developmental stage suggests protein function at

that stage

Northern Blotting

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Reverse transcriptase-polymerase chain reaction(RT-PCR)is quicker and more sensitive thanNorthern Blotting

Reverse transcriptase is added to mRNA to make

cDNA, cDNA serves as a template for PCR amplification of

the gene of interest

The products are run on a gel and the mRNA ofinterest identified

RT-PCR

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Fig. 20-13

TECHNIQUE

RESULTS

Gel electrophoresis

cDNAs

-globingene

PCR amplification

Embryonic stages

Primers

1 2 3 4 5 6

mRNAscDNA synthesis1

2

3

Reverse Transcriptase

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Cloning Animals: Nuclear Transplantation

Nuclear transplantation : Nucleus of an unfertilizedegg cell or zygote is replaced with the nucleus of a

differentiated cell

Experiments with frog embryos have shown that

a transplanted nucleus can often support normal

development of the egg

However, the older the donor nucleus, the lower the

percentage of normally developing tadpoles

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Is cloning an animal the same as cloning a gene?

NO

Cloning an animal = exact genetic

copy of that animal

Cloning a gene = exact copy of agene into a smaller piece of DNA e.g.

plasmid

Different types of cloning:

Artificial embryo twinning

SCNT

http://learn.genetics.utah.edu/content/tech/cloning/whatiscloning/

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Somatic cell nuclear transfer (SCNT)

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Oocyte Enucleation

http://learn.genetics.utah.edu/content/tech/cloning/whatiscloning/

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SCNT

http://learn.genetics.utah.edu/content/tech/cloning/whatiscloning/

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Fig. 20-17

EXPERIMENT

Less differ-entiated cell

RESULTS

Frog embryo Frog egg cell

UV

Donornucleustrans-planted

Frog tadpole

Enucleatedegg cell

Egg with donor nucleusactivated to begin

development

Fully differ-

entiated(intestinal) cell

Donornucleustrans-planted

Most develop

into tadpoles

Most stop developing

before tadpole stage

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Reproductive Cloning of Mammals

In 1997, Scottish researchers announced the birthof Dolly, a lamb cloned from an adult sheep by

nuclear transplantation from a differentiated

mammary cell

Dollys premature death in 2003, as well as her

arthritis, led to speculation that her cells were not

as healthy as those of a normal sheep, possibly

reflecting incomplete reprogramming of the

original transplanted nucleus

Fi 20 18

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Fig. 20-18

TECHNIQUE

Mammarycell donor

RESULTS

Surrogatemother

Nucleus frommammary cell

Culturedmammary cells

Implanted

in uterusof a thirdsheep

Early embryo

Nucleusremoved

Egg celldonor

Embryonicdevelopment

Lamb (Dolly)genetically identical to

mammary cell donor

Egg cellfrom ovary

Cells fused

Grown inculture

1

33

4

5

6

2

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Since 1997, cloning has been demonstrated inmany mammals, including mice, cats, cows, horses,

mules, pigs, and dogs

CC (for Carbon Copy) was the first cat cloned;

however, CC differed somewhat from her female

parent

Fi 20 19

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Fig. 20-19

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Problems Associated with Animal Cloning

Only a small percentage of cloned embryos developnormally to birth

Many epigenetic changes, such as

acetylation of histones or

methylation of DNA,

must be reversed in the nucleus from a donor animal

in order for genes to be expressed or repressed

appropriately for early stages of development

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Stem Cells of Animals

What is a stem cell? A stem cell is a relatively unspecializedcell that can

reproduceitself indefinitelyand differentiateinto

specialized cells of one or more types

Stem cells isolated from early embryos at the

blastocyst stage are called embryonic stem cells;

these are able to differentiate into all cell types

The adult body also has stem cells, which replace

non-reproducing specialized cells

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Culturedstem cells

Early human embryoat blastocyst stage

(mammalian equiva-lent of blastula)

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Blood cellsNerve cellsLiver cells

Cells generatingall embryoniccell types

Adult stem cells

Cells generatingsome cell types

Embryonic stem cells

From bone marrowin this example

The aim of stemcell research is tosupply cells for the

repair of damagedor diseased organs

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The practical applications of DNA

technology

Many fields benefit from DNA technology and

genetic engineering

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Medical Applications

Identification of human genes in which mutationplays a role in genetic diseases

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Diagnosis of Diseases

Diagnosis of many human genetic disorders byusing PCR

Primers corresponding to cloned disease genes areused.

Sequencing of the amplified product to identifydisease-causing mutation

Genetic disorders can also be tested for using

genetic markers that are linked to the disease-causing allele

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Single nucleotide polymorphisms (SNPs)are useful

genetic markers

These are single base-pair sites that vary in apopulation

When a restriction enzyme is added, SNPs result

in DNA fragments with different lengths, orrestriction fragment length polymorphism (RFLP)

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Disease-causing

allele

SNP

Normal allele

T

C

DNA

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Human Gene Therapy

Gene therapy is the alteration of an afflictedindividuals genes

Gene therapy holds great potential for treating

disorders traceable to a single defective gene

Vectors are used for delivery of genes into specific

types of cells, for example bone marrow

Gene therapy raises ethical questions, such as

whether human germ-line cells should be treated tocorrect the defect in future generations

Fig. 20-22

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g

Bone

marrow

Clonedgene

Bonemarrowcell frompatient

Insert RNA version of normal alleleinto retrovirus.

Retroviruscapsid

Viral RNA

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Inject engineered

cells into patient.

1

2

3

4

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Pharmaceutical Products

DNA technology and genetic research areimportant to the development of new drugs to

treat diseases

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The drug imatinib is a small molecule that inhibits

overexpression of a specific leukemia-causing

receptor

Pharmaceutical products that are proteins can be

synthesized on a large scale

Synthesis of Small Molecules for Use as Drugs

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Host cells in culture can be engineered to secrete aprotein as it is made

This is useful for the production of insulin, human

growth hormones, and vaccines

Protein Production in Cell Cultures

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Transgenic animals are made by introducing genes

from one species into the genome of another

animal

Transgenic animals are pharmaceutical factories,producers of large amounts of otherwise rare

substances for medical use

Pharm plants are also being developed to makehuman proteins for medical use

Protein Production by Pharm Animals and Plants