Chapter 20: Biotechnology

• 20.1 and 20.2 are the only sections we will cover in Chapter 20. The rest will be covered after the AP exam.

• Self Quiz Multiple Choice Questions p. 424 – 425 (1-8) and question 9

Overview: The DNA Toolbox

• Sequencing of the human genome was completed by 2007

• DNA sequencing has depended on advances in technology, starting with making recombinant DNAo Nucleotide sequences from two different sources, often

two species, are combined in vitro into the same DNA molecule

Genetic Engineering• Methods for making recombinant DNA are central

to genetic engineering, the direct manipulation of genes for practical purposes

• DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products

• An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

20.1 DNA cloning yields multiple copies of a gene or other DNA

segment

Cloning• To work directly with specific genes, scientists

prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning

Use of bacteria in cloning

• Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids – mainly E. coli

• Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosomeo Plasmid DNA may not always be needed for cell processes

• Cloned genes are useful for making copies of a particular gene and producing a protein product

Gene Cloning Using Bacteria

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

1. Foreign DNA is inserted into a plasmid2. The recombinant plasmid is inserted into a

bacterial cell3. Reproduction in the bacterial cell results in

cloning of the plasmid including the foreign DNA

• This results in the production of multiple copies of a single gene

Fig. 20-2a

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Plasmid

2

1

2

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

4

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

3

Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites

• A restriction enzyme usually makes many cuts, yielding restriction fragments

• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments

• DNA ligase

Fig. 20-3-1Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

53

35

1

Fig. 20-3-2Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

53

35

1

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

2

One possible combination

Fig. 20-3-3Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

53

35

1

One possible combination

Recombinant DNA molecule

DNA ligaseseals strands.

3

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

2

Cloning a Eukaryotic Gene in a Bacterial Plasmid

• In gene cloning, the original plasmid is called a cloning vectoro DNA molecule that can carry foreign DNA into a host cell and

replicate there• Bacterial plasmids are useful to clone

eukaryotic DNA:o Easy to isolate, manipulate and reintroduceo Multiply rapidly

Hummingbird β-globin Gene

CloningTurn to page 399. Summarize the steps in hummingbird β-globin gene cloning.

Fig. 20-4-1

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Fig. 20-4-2

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Fig. 20-4-3

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Bacteria carryingplasmids

Fig. 20-4-4

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Bacteria carryingplasmids

RESULTS

Colony carrying non-recombinant plasmidwith intact lacZ gene

One of manybacterialclones

Colony carrying recombinant plasmid with disrupted lacZ gene

Hummingbird β-globin Gene Cloning Follow Up Questions

1. What two genes were targeted in the hummingbird cloning experiment.

2. Where was the DNA cut? What enzyme was used to cut the DNA?

3. What enzyme was responsible for covalently bonding the “sticky ends”

4. Do all cells take up recombinant plasmids?5. What was included in the nutrient agar?6. Describe the experimental results.

Storing Cloned Genes in DNA Libraries

• A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome

• Plasmid clones contain copies of a particular gene within a plasmid

• A genomic library that is made using bacteriophages is stored as a collection of phage cloneso Normal infection process will produce many copies of the cloned DNA

Fig. 20-5

Bacterial clones

Recombinantplasmids

Recombinantphage DNA

or

Foreign genomecut up withrestrictionenzyme

(a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

Phageclones

Large plasmidLarge insertwith many genes

BACclone

Fig. 20-5a

Bacterial clones Recombinant

plasmids

Recombinantphage DNA

or

Foreign genomecut up withrestrictionenzyme

(a) Plasmid library (b) Phage library

Phageclones

BACs• A bacterial artificial chromosome (BAC) is a

large plasmid that has been trimmed down and can carry a large DNA insert

• BACs are another type of vector used in DNA library construction

cDNA• A complementary DNA (cDNA) library is made

by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell

• A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

Fig. 20-6-5

DNA innucleus

mRNAs in cytoplasm

Reversetranscriptase Poly-A tail

DNAstrand

Primer

mRNA

DegradedmRNA

DNA polymerase

cDNA

Screening a Library for Clones Carrying a Gene of Interest

• A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene

• This process is called nucleic acid hybridization

Expressing Cloned Eukaryotic Genes

• After a gene has been cloned, its protein product can be produced in larger amounts for research

• Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

Bacterial Expression Systems

• Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cellso Differences in promoter/control sequenceso Presence of introns

• To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter

• Use of cDNA can overcome issues with introns

Eukaryotic Cloning and Expression

Systems• To avoid gene expression problems

o Use of cultured eukaryotic cells as host cells• Yeasts and single-celled fungi – easy to grow, contain plasmids

o Yeast artificial chromosomes (YACs) • Behave like normal chromosomes in mitosis • Can carry more DNA than a plasmid

• Eukaryotic hosts can provide the post-translational modifications that many proteins require

Eukaryotic Cloning and Expression

Systems• One method of introducing recombinant DNA into

eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes

• Alternatively, scientists can inject DNA into cells using microscopically thin needles

• Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)• The polymerase chain reaction, PCR, can

produce many copies of a specific target segment of DNA

• A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

How does PCR work?• Heat denatures the DNA• Cooling allows hydrogen bonds to reform between

the strands of DNA• Heat-stable DNA polymerase base pairing from

target segment of DNA

Fig. 20-8a

5

Genomic DNA

TECHNIQUETargetsequence

3

3 5

Fig. 20-8b

Cycle 1yields

2molecules

Denaturation

Annealing

Extension

Primers

Newnucleo-tides

3 5

3

2

5 31

Fig. 20-8c

Cycle 2yields

4molecules

Fig. 20-8d

Cycle 3yields 8

molecules;2 molecules

(in whiteboxes)

match targetsequence

Quick Review• Define and distinguish between genomic libraries

using plasmids, phages, and cDNA• Describe the polymerase chain reaction (PCR)

and explain the advantages and limitations of this procedure

Concept 20.2: DNA technology allows us to

study the sequence, expression, and function

of a gene

Gel Electrophoresis• Pg. 405• Fragments of DNA (obtained through restriction

enzyme digestion) or PCR amplification are separated into bands based on physical differenceso Sizeo Electrical charge o Other physical properties

• Utilizes gel made of a polymer (usually polysaccharide) and an electrical field.

1. The gel is made using agarose (similar to gelatin, but made of seaweed), a buffer (salt water solution to allow electrical charges to flow through the gel), a flask, a microwave, the gel mold, and a gel comb. The agarose and buffer are mixed, microwaved, and poured into the gel mold.

2. The comb is placed in one end of the gel (to make the wells) and the gel cools.

3. To set up the electrophoresis box, buffer is poured into the box and the gel (still in the mold) is also placed in the box.

4. A loading buffer (to dye the DNA) is added to the DNA tube and the DNA samples are placed in each of the wells at the top of the gel using a pippetor, taking care not to break the gel. These wells can be seen as the darker rectangles at the top of the picture to the right.

1. Use a pippetor to place DNA size standard in the well next to the first well you placed the DNA in. This is used as a "ruler" to measure the DNA bands later.

2. An electric field is set up across the gel with the negative (-) end at the top where the wells are and the positive (+) end opposite the wells. When an electrical current is added the DNA will move towards the positive charge because DNA has a negative charge.

3. The short strands will move faster (and therefore farther) than the longer strands (due to some basic laws of physics) and strands of the same length will move the same distance. Thus, the DNA arranges itself on the gel according to length, longest strands being nearest to the starting wells.

4. The sorted DNA on the gel is stained with ethidium bromide so that it becomes visible when UV light is shown on the gel. The photo to the right represents a gel electrophoresis. You are not seeing individual strands of DNA, but rather groups of DNA of the same length (represented as the white bands).

5. The DNA strand lengths are measured in base pairs (bp) based on the DNA size standard bands.

• Work with a partner to summarize two of the DNA techniques.

• You will present your summary to the class.

Gene Sequence Determination

• Gel electrophoresis• Restriction fragment analysis• Southern blotting• Dideoxyribonucleotide chain termination method

Gene Expression Determination

• Northern blotting• Reverse transcriptase-polymerase chain reaction• In situ hybridization• DNA microarray assays

Gene Function Determination

• In vitro mutagenesis• RNA interference (covered in chapter 18)

Quick Review• Explain how gel electrophoresis is used to analyze

nucleic acids and to distinguish between two alleles of a gene

• Describe and distinguish between the Southern blotting procedure, Northern blotting procedure, and RT-PCR

Concept 20.3: Cloning organisms may lead to

production of stem cells for research and other

applications

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

Cloning Plants: Single-Cell Cultures

• One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism

• A totipotent cell is one that can generate a complete new organism

Cloning Animals: Nuclear Transplantation

• In nuclear transplantation, the nucleus of an unfertilized egg 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

Fig. 20-17

EXPERIMENT

Less differ-entiated cell

RESULTS

Frog embryo Frog egg cellUV

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

Reproductive Cloning of Mammals

• In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell

• Dolly’s 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

Fig. 20-18

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 ovaryCells fused

Grown inculture

1

33

4

5

6

2

Cloning Mammals• Since 1997, cloning has been demonstrated in

many 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”

Problems with Animal Cloning

• In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally 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

Stem Cells of Animals• A stem cell is a relatively unspecialized cell that

can reproduce itself indefinitely and differentiate into 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 nonreproducing specialized cells

Fig. 20-20

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 stem cell research is to supply cells for the repair of damaged or diseased organs

20.4 The practical applications of DNA

technology affect our lives in many ways

Practical Applications of DNA Technology

• Medical• Forensic• Environmental • Agricultural

o Transgenic animalso Genetically engineered plants

• Ti plasmid• Herbicidal resistance, pest resistance, improved nutritional value

Medical Applications • One benefit of DNA technology is identification of

human genes in which mutation plays a role in genetic diseases

Medical Applications• Diagnosis of Diseases

o Scientists can diagnose many human genetic disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation

o Genetic disorders can also be tested for using genetic markers that are linked to the disease-causing allele

• Single nucleotide polymorphisms (SNPs) are useful genetic markers

• These are single base-pair sites that vary in a population

• When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphism (RFLP)

Medical Applications• Human Gene Therapy

o Gene therapy is the alteration of an afflicted individual’s genes

o Gene therapy holds great potential for treating disorders traceable to a single defective gene

o Vectors are used for delivery of genes into specific types of cells, for example bone marrow

o Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

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.

1

2

3

4

• Pharmaceutical Productso DNA technology and genetic research are important to the

development of new drugs to treat diseases

Pharmaceutical Products

• Synthesis of Small Molecules for Use as Drugs• 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• Protein Production in Cell Culture

• Host cells in culture can be engineered to secrete a protein as it is made

• This is useful for the production of insulin, human growth hormones, and vaccines

Protein Production by “Pharm” Animals and Plants

• 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 make human proteins for medical use

Forensic Evidence and Genetic Profiles

• An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids

• Genetic profiles can be used to provide evidence in criminal and paternity cases and to identify human remains

• Genetic profiles can be analyzed using RFLP analysis by Southern blotting

• Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences

• PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths

• The probability that two people who are not identical twins have the same STR markers is exceptionally small

Fig. 20-24This photo shows EarlWashington just before his release in 2001,after 17 years in prison.

These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.

(a)

Semen on victim

Earl Washington

Source of sample

Kenneth Tinsley

STRmarker 1

STRmarker 2

STRmarker 3

(b)

17, 19

16, 18

17, 19

13, 16 12, 12

14, 15 11, 12

13, 16 12, 12

Environmental Clean Up

• Genetic engineering can be used to modify the metabolism of microorganisms

• Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

• Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels

Agricultural• Transgenic animals

• Genetically engineered plantso Ti plasmido Herbicidal resistance, pest resistance, improved nutritional value

Ethics• Potential benefits of genetic engineering must be

weighed against potential hazards of creating harmful products or procedures

• Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology

Concerns• Most public concern about possible hazards

centers on genetically modified (GM) organisms used as food

• Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives

Bioethics of Human Clonging and HeLa Cells