Chapter 12 DNA Technology February 27, 2013. DNA technology has led to advances in –creation of genetically modified crops and –identification and treatment

Chapter 12DNA Technology

February 27, 2013

• DNA technology has led to advances in

– creation of genetically modified crops and

– identification and treatment of genetic diseases

– criminology

Biology and Society: DNA, Guilt, and Innocence

© 2013 Pearson Education, Inc.

RECOMBINANT DNA TECHNOLOGY

• Biotechnology

– is the manipulation of organisms or their components to make useful products and

– has been used for thousands of years to

– make bread using yeast and

– selectively breed livestock for desired traits.


• DNA technology

– studying and manipulating genetic material,

– modifying specific genes

– moving genes between organisms



• Recombinant DNA = DNA from 2 sources combined to form a single DNA molecule.

• Genetic engineering = the direct manipulation of genes

• ex. By transferring the gene for a desired protein into a bacterium or yeast, proteins that are naturally present in only small amounts can be produced in large quantities.



Making Humulin

• In 1982, the world’s first genetically engineered pharmaceutical product was sold.

• Humulin = human insulin

– produced by genetically modified bacteria

– used today by 4 million+ people with

– diabetes.


Figure 12.3

• Also makes

– HGH = human growth hormone

– EPO = erythropoietin

– Vaccines

Making Humulin


Genetically Modified (GM) Foods

• Genetically modified organisms (GMO’s) = organisms that have one or more genes by artificial means.

• Transgenic = organism with a gene from another species.


• In the USA, ~ ½ of the corn crop and ¾ + of the soybean and cotton crops are GM.



• GM Strawberry have bacterial proteins = natural antifreeze

• GM Potatoes and rice produce harmless proteins; may one day serve as edible vaccines.



• “Golden rice 2”– transgenic rice with genes from daffodils and corn– could help prevent vitamin A deficiency and

resulting blindness.



Golden Rice Debate

http://sciencecases.lib.buffalo.edu/cs/files/golden_rice_debate.pdf

“Pharm” Animals

• No transgenic animals are sold as food (yet).

• GM pig has gene for human hemoglobin, which can be used in blood transfusions.

• GM pigs can produce proteins that convert “bad” fatty acids to omega-3 fatty acids.


How are transgenic organisms created?

Recombinant DNA Techniques

• Plasmids = workhorses of modern biotechnology

• small, circular DNA molecules

• replicate separately from the larger bacterial chromosome.

• can carry virtually any gene

• act as vectors = move genes from one cell to another


Figure 12.7

Plasmids

Bacterialchromosome

Remnant ofbacterium

Co

lori

zed

TE

M

Figure 12.8a

Plasmid

Bacterial cell

Isolate plasmids.

Cut both DNAswith same enzyme.

Isolate DNA.

Geneof

interestOthergenes

DNA fragmentsfrom cell

Mix the DNA fragments and join them together.

DNA

Cell containingthe gene ofinterest

Gene of interest

Recombinant DNA plasmids

1 2

3

4

Figure 12.8b

5

6

7

Bacteria take up recombinant plasmids.

Recombinant bacteriaBacterial clone

Clone the bacteria.

Find the clone with the gene of interest.

Figure 12.8c

8

Protein fordissolvingclots

Protein for“stone-washing” jeans

Harvestedproteinsmay beused directly.

The geneand proteinof interestare isolatedfrom thebacteria.

Genes maybe insertedinto otherorganisms.

Some usesof genes

Some usesof proteins

Gene for pestresistance

Genes for cleaning up toxic waste

Figure 12.8

Plasmid

Bacterial cellIsolate plasmids.1 2

3

4

5

6

7

Cut both DNAswith same enzyme.

Isolate DNA.

Geneof

interestOthergenes

DNA fragmentsfrom cell

DNA

Cell containingthe gene of interest

Mix the DNA fragments and join them together.

Gene of interest

Recombinant DNA plasmids

Bacteria take up recombinant plasmids.

Recombinant bacteriaBacterial clone

Clone the bacteria.

Find the clone with the gene of interest.

A protein is used todissolve blood clotsin heart attacktherapy.

A protein is used to prepare“stone-washed” blue jeans.

Bacteriaproduceproteins,which can be harvestedand used directly.

The geneand proteinof interestare isolatedfrom thebacteria.

Genes maybe insertedinto otherorganisms.

Some usesof genes

Some usesof proteins

A gene for pestresistance isinserted intoplants.

A gene is used to alterbacteria for cleaningup toxic waste.

8

A Closer Look: Cutting and Pasting DNA with Restriction Enzymes

• Recombinant DNA is produced by combining two ingredients:

1. a bacterial plasmid and

2. the gene of interest.

• To combine these ingredients, a piece of DNA must be spliced into a plasmid.


• DNA gets spliced by

– using restriction enzymes, which cut DNA at specific restriction sites

– Producing restriction fragments with “sticky ends”

– DNA ligase connects the DNA fragments into one piece by forming new bonds

A Closer Look: Cutting and Pasting DNA with Restriction Enzymes


Figure 12.9-1

Recognition site (recognition sequence)for a restriction enzyme

Restrictionenzyme

Sticky end

Sticky end

DNA

A restriction enzyme cuts the DNA into fragments.

1

Figure 12.9-2


Restrictionenzyme

Sticky end

Sticky end

DNA

A DNA fragment is added from another source.


1

2

Figure 12.9-3


Restrictionenzyme

Sticky end

Sticky end

DNA



Fragments stick together bybase pairing.

1

2

3

Figure 12.9-4


Restrictionenzyme

Sticky end

Sticky end

DNA

DNAligase

Recombinant DNA molecule



Fragments stick together bybase pairing.

DNA ligase joins the fragments into strands.

1

2

3

4

How is DNA used in forensics?

Biology and Society: DNA, Guilt, and Innocence

• DNA profiling = analysis of DNA samples to determine whether the samples come from the same individual.


Figure 12.13-1

DNA isolated Crime scene Suspect 1 Suspect 21

Figure 12.13-2

DNA isolated

DNA amplified

Crime scene Suspect 1 Suspect 21

2

Figure 12.13-3

DNA isolated

DNA amplified

DNA compared


2

3

Investigating Murder, Paternity, and Ancient DNA

• DNA profiling can be used to

– Test guilt of suspected criminals,

– identify tissue samples of victims

– resolve paternity cases

– identify contraband animal products

– trace the evolutionary history of organisms


NAU’s Innocence Project

Arizona Innocence Project

http://azdailysun.com/news/local/convicted-but-not-guilty/article_9745416d-261f-581a-9230-1bc25676ef41.html

http://arizonainnocenceproject.org/AzIP_4/Home.html

Figure 12.14

DNA Profiling Techniques The Polymerase Chain Reaction (PCR)

• The polymerase chain reaction (PCR)

– quickly copies a specific segment of DNA

– generates enough DNA, from even minute amounts of blood or other tissue, to allow DNA profiling.


Figure 12.15

InitialDNAsegment

Number of DNA molecules1 2 4 8

• Short tandem repeats (STRs) are

– short sequences of DNA

– repeated many times together in the genome

• STR analysis

– method of DNA profiling

– Compares lengths of STR sequences

Short Tandem Repeat (STR) Analysis


Figure 12.16

Crime scene DNA

Suspect’s DNA

Same number ofshort tandem repeats

Different numbers ofshort tandem repeats

STR site 1 STR site 2

AGAT

AGAT GATA

GATA

Figure 12.17-1

Mixture of DNAfragments ofdifferent sizes

Powersource

Gel electrophoresis = method for sorting DNA fragments by size

Figure 12.17-2


Powersource

Figure 12.17-3

Band of longest(slowest) fragments

Band of shortest(fastest) fragments


Powersource

Figure 12.18

Amplifiedcrime sceneDNA

Amplifiedsuspect’sDNA

Longerfragments

Shorterfragments

Figure 12.UN02


DNA

Polymerase chainreaction (PCR)amplifies STRsites

LongerDNAfragments

ShorterDNAfragments

DNA fragments compared by gel electrophoresis

Gel

(Bands of shorter fragments move faster toward the positive pole.)

Gene Therapy = giving “good” gene to patientthat lacks it.

History of Gene Therapy

http://history.nih.gov/exhibits/genetics/sect4.htm

Figure 12.UN03

RNA versionof a normalhuman gene

Virus withRNA genome

Bonemarrow

A normal human gene is transcribedand translated in a patient, potentiallycuring the genetic disease permanently

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Chapter 12 DNA Technology February 27, 2013. DNA technology has led to advances in –creation of genetically modified crops and –identification and treatment