Biotechnology DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid...

Biotechnology

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Plasmid

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Gene cloning involves using bacteria to make multiple copies of a gene

DNA Cloning: An Overview

Fig. 20-2b

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Gene ofInterest

Protein expressedby gene of interest

Basic research andvarious applications

Copies of gene Protein harvested

Basicresearchon gene

Basicresearchon protein

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Recombinantbacterium

Gene for pest resistance inserted into plants

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

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Restriction Enzymes

• Gene cloning was made possible by the discovery of restriction enzymes.

• Many different enzymes exist– named after the organism in which they are found

• EcoRI (E. coli), HindIII (Haemophilus influenza), PstI (Providencia stuartii)

Fig. 20-3-1Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

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Fig. 20-3-2Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

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DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

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One possible combination

Fig. 20-3-3Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

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One possible combination

Recombinant DNA molecule

DNA ligaseseals strands.

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DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

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Fig. 20-UN3

Cut by same restriction enzyme,mixed, and ligated

DNA fragments from genomic DNAor cDNA or copy of DNA obtainedby PCR

Vector

Recombinant DNA plasmids

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Genomic DNA

TECHNIQUE

Cycle 1yields

2molecules

Denaturation

Annealing

Extension

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in whiteboxes)

match targetsequence

Targetsequence

Primers

Newnucleo-tides

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Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)

PCR can make billions of a specific DNA segment ina few hours.

PCR produces many copies of a specific target segment of DNA

PCR Animation

PC

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NA

Am

plifi

catio

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PCR Applications

30,000 y. o. woolly mammoth

In forensics, PCR requires only smallsamples of DNA to analyze

DNA polymerase (Taq)

Fig. 20-9

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Shortermolecules

Gel

AnodeCathode

TECHNIQUE

RESULTS

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+

–

–

One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis

Gel Electrophoresis

TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT

human genome

Fig. 20-12

DNA(template strand)

TECHNIQUE

RESULTS

DNA (template strand)

DNA polymerase

Primer Deoxyribonucleotides

Shortest

Dideoxyribonucleotides(fluorescently tagged)

Labeled strands

Longest

Shortest labeled strand

Longest labeled strand

Laser

Directionof movementof strands

Detector

Last baseof longest

labeledstrand

Last baseof shortest

labeledstrand

dATP

dCTP

dTTP

dGTP

ddATP

ddCTP

ddTTP

ddGTP

DNA Sequencing

Relatively short DNA fragmentscan be sequenced using the dideoxychain termination method.

Sequencing Video

DN

A S

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ncin

g

DN

A S

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ncer

New approaches have accelerated the pace of genome sequencing

The Human Genome Project was proposed in 1986 to determine the normal sequence of all human DNA.

The history of sequencing• New Generation Sequencing

Millions of different fragments are sequenced at the same time. This is called massively parallel sequencing.

50 µm

Studying the Expression of Interacting Groups of Genes

DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions Microarray Video

From: National Academy of Science, 2009

Metagenomics

Genetic diversity is explored without isolating intact organisms.

• Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell

Cloning organisms has the potential to generate stem cells for research

Fig. 20-16

EXPERIMENT

Transversesection ofcarrot root

2-mgfragments

Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid.

Singlecellsbegan todivide.

Embryonicplant developedfrom a culturedsingle cell.

Plantlet wascultured onagar medium andlater, plantedin soil.

A singlesomaticcarrot celldevelopedinto a maturecarrot plant.

RESULTS

Can a differentiated plant cell develop into a whole plant?

Fig. 20-17

EXPERIMENT

Less differ-entiated cell

RESULTS

Frog embryo Frog egg cell

UV

Donornucleustrans-planted

Frog tadpole

Enucleated egg cell

Egg with donor nucleus activated to begin

development

Fully differ-entiated(intestinal) cell

Donor nucleus trans-planted

Most developinto tadpoles

Most stop developingbefore tadpole stage

Can the nucleus from a differentiated animal cell direct development of an organism?

TECHNIQUE

Mammarycell donor

RESULTS

Surrogatemother

Nucleus frommammary cell

Culturedmammary cells

Implantedin uterusof a thirdsheep

Early embryo

Nucleusremoved

Egg celldonor

Embryonicdevelopment Lamb (“Dolly”)

genetically identical tomammary cell donor

Egg cellfrom ovary

Cells fused

Grown inculture

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Reproductive cloning of a mammal by nuclear transplantation

In 1997, Scottish researchers announced the birth of Dolly

Fig. 20-19

CC (for Carbon Copy) was the first cat cloned

The practical applications of DNA technology

• Many fields benefit from DNA technology and genetic engineering

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

A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types

Stem cell animation

Nuclear implantation

Fig. 20-22

Bonemarrow

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 engineeredcells into patient.

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Gene therapy

Fig. 20-25

Site whererestrictionenzyme cuts

T DNA

Plant with new trait

Tiplasmid

Agrobacterium tumefaciens

DNA withthe geneof interest

RecombinantTi plasmid

TECHNIQUE

RESULTS

Genetic engineering in plants has been used to transfer many useful genes

Plant Breeding compared to Genetic Modification of Plants

GHOSTS

Fig. 20-11TECHNIQUE

Nitrocellulosemembrane (blot)

Restrictionfragments

Alkalinesolution

DNA transfer (blotting)

Sponge

Gel

Heavyweight

Papertowels

Preparation of restriction fragments Gel electrophoresis

I II III

I II IIII II III

Radioactively labeledprobe for -globin gene

DNA + restriction enzyme

III HeterozygoteII Sickle-cellallele

I Normal-globinallele

Film overblot

Probe detectionHybridization with radioactive probe

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globin allele

Probe base-pairswith fragments

Nitrocellulose blot

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