_12_Lecture_Presentation.ppt

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PowerPoint Lectures for

Campbell Biology: Concepts & Connections, Seventh Edition Reece, Taylor, Simon, and Dickey

Chapter 12

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

DNA technology

– has rapidly revolutionized the field of forensics,

– permits the use of gene cloning to produce medical and industrial products,

– allows for the development of genetically modified organisms for agriculture,

– _{permits the investigation of historical questions about}

human family and evolutionary relationships, and

– is invaluable in many areas of biological research.

Introduction

(3)

Figure 12.0_1

Chapter 12: Big Ideas

Gene Cloning

DNA Profiling

Genetically Modified Organisms

(4)

(5)

GENE CLONING

(6)

12.1 Genes can be cloned in recombinant plasmids



Biotechnology

is the manipulation of organisms

or their components to make useful products.



For thousands of years, humans have

– used microbes to make wine and cheese and

– selectively bred stock, dogs, and other animals.



DNA technology

is the set of modern techniques

used to study and manipulate genetic material.

(7)

(8)

12.1 Genes can be cloned in recombinant plasmids



Genetic engineering

involves manipulating

genes for practical purposes.

– Gene cloning leads to the production of multiple,

identical copies of a gene-carrying piece of DNA.

– Recombinant DNA is formed by joining nucleotide

sequences from two different sources.

– One source contains the gene that will be cloned.

– Another source is a gene carrier, called a vector.

– Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors.

(9)



Steps in cloning a gene

1. Plasmid DNA is isolated.

2. DNA containing the gene of interest is isolated.

3. Plasmid DNA is treated with a restriction enzyme that cuts in one place, opening the circle.

4. DNA with the target gene is treated with the same enzyme and many fragments are produced.

5. Plasmid and target DNA are mixed and associate with each other.

12.1 Genes can be cloned in recombinant plasmids

(10)

6. Recombinant DNA molecules are produced when

DNA ligase joins plasmid and target segments

together.

7. The recombinant plasmid containing the target gene is taken up by a bacterial cell.

8. The bacterial cell reproduces to form a clone, a group of genetically identical cells descended from a single ancestral cell.

12.1 Genes can be cloned in recombinant plasmids

(11)

Figure 12.1B

E. coli bacterium

Bacterial

chromosome A plasmidis isolated.

Gene of interest The plasmid is cut with an enzyme. Plasmid

The cell’s DNA is isolated.

The cell’s DNA is cut with the same enzyme.

DNA

Examples of gene use A cell with DNA

containing the gene of interest

Gene of interest

The targeted fragment and plasmid DNA are combined.

DNA ligase is added, which joins the two DNA molecules.

Gene of interest

Genes may be inserted into other organisms.

The recombinant plasmid is taken up by a bacterium through transformation.

Examples of protein use

(12)

Figure 12.1B_s1

E. coli

bacterium

Bacterial chromosome

A plasmid is isolated.

Gene of interest Plasmid

DNA A cell with DNA containing the gene of interest

(13)

Figure 12.1B_s2 E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest Plasmid

DNA A cell with DNA containing the gene of interest

1

3

2

4

The plasmid is cut with an enzyme.

The cell’s DNA is cut with the same enzyme. Gene

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DNA A cell with DNA containing the gene of interest 1 3 2 4 5

The plasmid is cut with an enzyme.

The cell’s DNA is cut with the same enzyme. Gene

of interest

(15)

DNA A cell with DNA containing the gene of interest 1 3 2 4 5 6

The plasmid is cut with an enzyme.

The cell’s DNA is cut with the same enzyme. Gene

of interest

(16)

Figure 12.1B_s5

Gene

of interest

The recombinant plasmid is taken up by a bacterium through transformation. Recombinant

bacterium Recombinant DNA

plasmid

(17)

Figure 12.1B_s6

Gene

of interest

The recombinant plasmid is taken up by a bacterium through transformation.

The bacterium reproduces.

Clone of cells

Recombinant bacterium Recombinant DNA

plasmid

7

(18)

Figure 12.1B_s7

Gene

of interest

The recombinant plasmid is taken up by a bacterium through transformation. Harvested proteins may be used directly. The bacterium reproduces. Clone of cells Recombinant bacterium Recombinant DNA plasmid

Genes may be inserted into other organisms.

9 7

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(22)

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12.2 Enzymes are used to “cut and paste” DNA



Restriction enzymes

cut DNA at specific

sequences.

– Each enzyme binds to DNA at a different restriction site.

– Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends.”

– Fragments with complementary sticky ends can

associate with each other, forming recombinant DNA.



DNA ligase joins DNA fragments together.

(24)

Figure 12.2_s1

A restriction enzyme cuts the DNA into fragments.

Restriction enzyme recognition sequence Restriction

enzyme Sticky

end Sticky

end DNA

1

(25)

Figure 12.2_s2

A restriction enzyme cuts the DNA into fragments. Restriction enzyme recognition sequence Restriction enzyme Gene of interest A DNA fragment

from another source is added.

(26)

Figure 12.2_s3

A restriction enzyme cuts the DNA into fragments. Restriction enzyme recognition sequence Restriction enzyme Gene of interest A DNA fragment

(27)

Figure 12.2_s4

A restriction enzyme cuts the DNA into fragments. Restriction enzyme recognition sequence Restriction enzyme Gene of interest A DNA fragment

(28)

12.3 Cloned genes can be stored in genomic

libraries



A

genomic library

is a collection of all of the

cloned DNA fragments from a target genome.



Genomic libraries can be constructed with

different types of vectors:

– plasmid library: genomic DNA is carried by plasmids,

– bacteriophage (phage) library: genomic DNA is incorporated into bacteriophage DNA,

– bacterial artificial chromosome (BAC) library: specialized plasmids that can carry large DNA sequences.

(29)

Figure 12.3

A genome is cut up with a restriction enzyme

Recombinant phage DNA Recombinant

plasmid

Bacterial

clone Phageclone or

(30)

12.4 Reverse transcriptase can help make genes

for cloning



Complementary DNA

(

cDNA

) can be used to

clone eukaryotic genes.

– In this process, mRNA from a specific cell type is the template.

– Reverse transcriptase produces a DNA strand from

mRNA.

– DNA polymerase produces the second DNA strand.

(31)

12.4 Reverse transcriptase can help make genes

for cloning



Advantages of cloning with cDNA include the

ability to

– study genes responsible for specialized characteristics of a particular cell type and

– obtain gene sequences

– that are smaller in size,

– easier to handle, and

– do not have introns.

(32)

Figure 12.4

CELL NUCLEUS

DNA of a eukaryotic gene

RNA

transcript

mRNA

TEST TUBE

Reverse transcriptase cDNA strand

being synthesized

Direction of synthesis

Breakdown of RNA

Synthesis of second DNA strand

Isolation of mRNA from the cell and the addition of reverse transcriptase; synthesis of a DNA strand

cDNA of gene (no introns)

Exon Intron Exon Intron Exon

Transcription

RNA splicing (removes introns and joins exons)

1

2

3

4

(33)

Figure 12.4_1

CELL NUCLEUS

DNA of a eukaryotic gene

RNA

transcript

mRNA

Exon Intron

Transcription

RNA splicing (removes introns and joins exons)

1

2

(34)

Figure 12.4_2

TEST TUBE

Reverse transcriptase

cDNA strand

being synthesized

Direction of synthesis

Breakdown of RNA

Synthesis of second DNA strand

Isolation of mRNA from the cell and the addition of reverse transcriptase; synthesis of a DNA strand

cDNA of gene (no introns)

3

4

(35)

12.5 Nucleic acid probes identify clones carrying

specific genes



Nucleic acid probes

bind very selectively to

cloned DNA.

– Probes can be DNA or RNA sequences

complementary to a portion of the gene of interest.

– A probe binds to a gene of interest by base pairing.

– Probes are labeled with a radioactive isotope or fluorescent tag for detection.

(36)

12.5 Nucleic acid probes identify clones carrying

specific genes



One way to screen a gene library is as follows:

1. Bacterial clones are transferred to filter paper.

2. Cells are broken apart and the DNA is separated into single strands.

3. A probe solution is added and any bacterial colonies carrying the gene of interest will be tagged on the filter paper.

4. The clone carrying the gene of interest is grown for further study.

(37)

Figure 12.5

Radioactive nucleic acid probe (single-stranded DNA)

Base pairing highlights the gene of interest. The probe is mixed with single-stranded DNA from a genomic library. Single-stranded

(38)

GENETICALLY MODIFIED

ORGANISMS

(39)

12.6 Recombinant cells and organisms can

mass-produce gene products



Recombinant cells and organisms constructed by

DNA technologies are used to manufacture many

useful products, chiefly proteins.



Bacteria are often the best organisms for

manufacturing a protein product because bacteria

– have plasmids and phages available for use as gene-cloning vectors,

– can be grown rapidly and cheaply,

– can be engineered to produce large amounts of a particular protein, and

– often secrete the proteins directly into their growth medium.

(40)

12.6 Recombinant cells and organisms can



Yeast cells

– are eukaryotes,

– have long been used to make bread and beer,

– can take up foreign DNA and integrate it into their genomes,

– _{have plasmids that can be used as gene vectors, and} – are often better than bacteria at synthesizing and

secreting eukaryotic proteins.

(41)

12.6 Recombinant cells and organisms can



Mammalian cells must be used to produce

proteins with chains of sugars. Examples include

– human erythropoietin (EPO), which stimulates the production of red blood cells,

– factor VIII to treat hemophilia, and

– tissue plasminogen activator (TPA) used to treat heart attacks and strokes.

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(43)

(44)

(45)

12.6 Recombinant cells and organisms can



Pharmaceutical researchers are currently exploring

the mass production of gene products by

– whole animals or

– plants.



Recombinant animals

– are difficult and costly to produce and

– must be cloned to produce more animals with the same traits.

(46)

(47)

(48)

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(50)

12.7 CONNECTION: DNA technology has

changed the pharmaceutical industry and

medicine



Products of DNA technology are already in use.

– Therapeutic hormones produced by DNA technology include

– insulin to treat diabetes and

– human growth hormone to treat dwarfism.

– DNA technology is used to

– test for inherited diseases,

– detect infectious agents such as HIV, and

– produce vaccines, harmless variants (mutants) or derivatives of a pathogen that stimulate the immune system.

(51)

(52)

(53)

12.8 CONNECTION: Genetically modified

organisms are transforming agriculture



Genetically modified

(

GM

) organisms contain

one or more genes introduced by artificial means.



Transgenic organisms

contain at least one gene

from another species.

(54)

12.8 CONNECTION: Genetically modified



The most common vector used to introduce new

genes into plant cells is

– a plasmid from the soil bacterium Agrobacterium

tumefaciens and

– called the Ti plasmid.

(55)

Figure 12.8A_s1

Restriction site

The gene is inserted into the plasmid.

Recombinant Ti plasmid

DNA containing the gene for a desired trait

1

Ti plasmid

(56)

Figure 12.8A_s2

Restriction site

The recombinant plasmid is

introduced into

a plant cell. _{DNA carrying} the new gene

Recombinant Ti plasmid

Plant cell DNA containing the

gene for a desired trait

(57)

Figure 12.8A_s3

Restriction site

The recombinant plasmid is

introduced into a plant cell.

The plant cell grows into a plant.

DNA carrying the new gene

A plant with the new trait Recombinant Ti plasmid Plant cell DNA containing the

gene for a desired trait

(58)

12.8 CONNECTION: Genetically modified



GM plants are being produced that

– are more resistant to herbicides and pests and

– provide nutrients that help address malnutrition.



GM animals are being produced with improved

nutritional or other qualities.

(59)

(60)

12.9 Genetically modified organisms raise

concerns about human and environmental

health



Scientists use safety measures to guard against

production and release of new pathogens.



Concerns related to GM organisms include the

potential

– introduction of allergens into the food supply and

– spread of genes to closely related organisms.



Regulatory agencies are trying to address the

– safety of GM products,

– labeling of GM produced foods, and

– safe use of biotechnology.

(61)

(62)

(63)

12.10 CONNECTION: Gene therapy may

someday help treat a variety of diseases



Gene therapy

aims to treat a disease by

supplying a functional allele.



One possible procedure is the following:

1. Clone the functional allele and insert it in a retroviral vector.

2. Use the virus to deliver the gene to an affected cell type from the patient, such as a bone marrow cell.

3. Viral DNA and the functional allele will insert into the patient’s chromosome.

4. Return the cells to the patient for growth and division.

(64)

12.10 CONNECTION: Gene therapy may



Gene therapy

is an

– alteration of an afflicted individual’s genes and

– attempt to treat disease.



Gene therapy may be best used to treat disorders

traceable to a single defective gene.

(65)

Figure 12.10

An RNA version of a normal human gene is inserted into a retrovirus. RNA genome of virus Retrovirus

Bone marrow cells are infected with the virus.

Viral DNA carrying the human gene inserts into the cell’s chromosome. Bone marrow

cell from the patient

Bone marrow The engineered

cells are injected into the patient.

Cloned gene

(normal allele) 1

2

3

(66)

Figure 12.10_1

An RNA version of a normal human gene is inserted into a retrovirus.

RNA genome of virus

Retrovirus Cloned gene

(67)

Figure 12.10_2

Bone marrow cells are infected with the virus.

Viral DNA carrying the human gene inserts into the cell’s chromosome.

Bone marrow

cell from the patient

Bone marrow The engineered

cells are injected into the patient.

2

3

(68)

12.10 CONNECTION: Gene therapy may



The first successful human gene therapy trial in

2000

– tried to treat ten children with SCID (severe combined immune deficiency),

– helped nine of these patients, but

– caused leukemia in three of the patients, and

– resulted in one death.

(69)

12.10 CONNECTION: Gene therapy may



The use of gene therapy raises many questions.

– How can we build in gene control mechanisms that make appropriate amounts of the product at the right time and place?

– How can gene insertion be performed without harming other cell functions?

– Will gene therapy lead to efforts to control the genetic makeup of human populations?

– Should we try to eliminate genetic defects in our children and descendants when genetic variety is a necessary ingredient for the survival of a species?

(70)

DNA PROFILING

(71)

12.11 The analysis of genetic markers can

produce a DNA profile



DNA profiling

is the analysis of DNA fragments to

determine whether they come from the same

individual. DNA profiling

– compares genetic markers from noncoding regions that show variation between individuals and

– involves amplifying (copying) of markers for analysis.

(72)

Figure 12.11

DNA is isolated.

1

2

3

The DNA of selected markers is amplified.

The amplified DNA is

compared.

(73)

12.12 The PCR method is used to amplify DNA

sequences



Polymerase chain reaction

(

PCR

) is a method of

amplifying a specific segment of a DNA molecule.



PCR relies upon a pair of

primers

that are

– short,

– chemically synthesized, single-stranded DNA molecules, and

– complementary to sequences at each end of the target sequence.



PCR

– is a three-step cycle that

– doubles the amount of DNA in each turn of the cycle.

(74)

Figure 12.12

Cycle 1

yields two molecules yields four moleculesCycle 2

DNA polymerase adds nucleotides. Primers bond with ends of target sequences. Heat separates DNA strands. Genomic DNA Target sequence

Primer New DNA

Cycle 3

yields eight molecules

3

5

3

5 5

5

3 3

(75)

Figure 12.12_1

Cycle 1

yields two molecules

DNA polymerase adds nucleotides. Primers bond with ends of target sequences. Heat separates DNA strands. Genomic DNA Target sequence

Primer New DNA

5

3

5

3 3 5

5

5 3 5

5 3

5

3 3

5 5 3

5 3

3 2

(76)

Figure 12.12_2

Cycle 2

(77)



The advantages of PCR include

– the ability to amplify DNA from a small sample,

– obtaining results rapidly, and

– a reaction that is highly sensitive, copying only the target sequence.

12.12 The PCR method is used to amplify DNA

sequences

(78)

12.13 Gel electrophoresis sorts DNA molecules

by size



Gel electrophoresis

can be used to separate

DNA molecules based on size as follows:

1. A DNA sample is placed at one end of a porous gel.

2. Current is applied and DNA molecules move from the negative electrode toward the positive electrode.

3. Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel.

4. DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence.

5. Each band is a collection of DNA molecules of the same length.

(79)

Figure 12.13

A mixture of DNA fragments of different sizes

Power source

Gel

Completed gel

(80)

Figure 12.13_1

A mixture of DNA fragments of

different sizes

Power source

Gel

Completed gel

Longer (slower) molecules

(81)

(82)

12.14 STR analysis is commonly used for DNA

profiling



Repetitive DNA

consists of nucleotide sequences

that are present in multiple copies in the genome.



Short tandem repeats

(

STRs

) are short nucleotide

sequences that are repeated in tandem,

– composed of different numbers of repeating units in individuals and

– _{used in DNA profiling.}



STR analysis

– compares the lengths of STR sequences at specific sites in the genome and

– typically analyzes 13 different STR sites.

(83)

Figure 12.14A

Crime scene DNA

Suspect’s DNA

STR site 1 STR site 2

The number of short

(84)

Figure 12.14B

Crime scene DNA

Suspect’s DNA

Longer STR fragments

(85)

12.15 CONNECTION: DNA profiling has provided

evidence in many forensic investigations



DNA profiling is used to

– determine guilt or innocence in a crime,

– settle questions of paternity,

– identify victims of accidents, and

– probe the origin of nonhuman materials.

(86)

(87)

(88)

12.16 RFLPs can be used to detect differences in

DNA sequences



A

single nucleotide polymorphism

(

SNP

) is a

variation at a single base pair within a genome.



Restriction fragment length polymorphism

(

RFLP

) is a change in the length of restriction

fragments due to a SNP that alters a restriction

site.



RFLP analysis involves

– producing DNA fragments by restriction enzymes and

– sorting these fragments by gel electrophoresis.

(89)

Figure 12.16

Restriction enzymes

added

DNA sample 1 DNA sample 2

Cut Cut Cut w x y y z Sample

1 Sample2

(90)

Figure 12.16_1

Restriction enzymes

added

DNA sample 1 DNA sample 2

w

x

y y

z Cut

(91)

Figure 12.16_2

Sample 1

z

x

w

y y

Longer

fragments

Shorter fragments

(92)

GENOMICS

(93)

12.17 Genomics is the scientific study of whole

genomes



Genomics

is the study of an organism’s complete

set of genes and their interactions.

– Initial studies focused on prokaryotic genomes.

– Many eukaryotic genomes have since been investigated.

(94)

(95)

12.17 Genomics is the scientific study of whole

genomes



Genomics allows another way to examine

evolutionary relationships.

– Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans.

– Functions of human disease-causing genes have been determined by comparing human genes to similar

genes in yeast.

(96)

12.18 CONNECTION: The Human Genome

Project revealed that most of the human

genome does not consist of genes



The goals of the

Human Genome Project

(

HGP

)

included

– determining the nucleotide sequence of all DNA in the human genome and

– identifying the location and sequence of every human gene.

(97)

12.18 CONNECTION: The Human Genome

Project revealed that most of the human

genome does not consist of genes



Results of the Human Genome Project indicate that

– humans have about 20,000 genes in 3.2 billion nucleotide pairs,

– only 1.5% of the DNA codes for proteins, tRNAs, or rRNAs, and

– the remaining 98.5% of the DNA is noncoding DNA including

– telomeres, stretches of noncoding DNA at the ends of chromosomes, and

– transposable elements, DNA segments that can move or be copied from one location to another within or between

chromosomes.

(98)

Figure 12.18

Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%)

(99)

12.19 The whole-genome shotgun method of

sequencing a genome can provide a wealth

of data quickly



The Human Genome Project proceeded through

three stages that provided progressively more

detailed views of the human genome.

1. A low-resolution linkage map was developed using RFLP analysis of 5,000 genetic markers.

2. A physical map was constructed from nucleotide

distances between the linkage-map markers.

3. DNA sequences for the mapped fragments were determined.

(100)

12.19 The whole-genome shotgun method of

of data quickly



The

whole-genome shotgun method

– was proposed in 1992 by molecular biologist J. Craig Venter, who

– used restriction enzymes to produce fragments that were cloned and sequenced in just one stage and

– ran high-performance computer analyses to assemble the sequence by aligning overlapping regions.

(101)

12.19 The whole-genome shotgun method of

of data quickly



Today, this whole-genome shotgun approach is

the method of choice for genomic researchers

because it is

– relatively fast and

– inexpensive.



However, limitations of the whole-genome shotgun

method suggest that a hybrid approach using

genome shotgunning and physical maps may

prove to be the most useful.

(102)

Figure 12.19

Chromosome

Chop up each chromosome with restriction enzymes

Sequence the fragments DNA fragments

Align the fragments

(103)

12.20 Proteomics is the scientific study of the full

set of proteins encoded by a genome



Proteomics

– is the study of the full protein sets encoded by genomes and

– investigates protein functions and interactions.



The human proteome includes about 100,000

proteins.



Genomics and proteomics are helping biologists

study life from an increasingly holistic approach.

(104)

12.21 EVOLUTION CONNECTION: Genomes

hold clues to human evolution



Human and chimp genomes differ by

– 1.2% in single-base substitutions and

– 2.7% in insertions and deletions of larger DNA sequences.



Genes showing rapid evolution in humans include

– genes for defense against malaria and tuberculosis,

– a gene regulating brain size, and

– the FOXP2 gene involved with speech and vocalization.

(105)

12.21 EVOLUTION CONNECTION: Genomes

hold clues to human evolution



Neanderthals

– were close human relatives,

– were a separate species,

– also had the FOXP2 gene,

– may have had pale skin and red hair, and

– were lactose intolerant.

(106)

(107)

1.

Explain how plasmids are used in gene cloning.

2.

Explain how restriction enzymes are used to “cut

and paste” DNA into plasmids.

3.

Explain how plasmids, phages, and BACs are used

to construct genomic libraries.

4.

Explain how a cDNA library is constructed and how

it is different from genomic libraries constructed

using plasmids or phages.

5.

Explain how a nucleic acid probe can be used to

identify a specific gene.

You should now be able to

(108)

6.

Explain how different organisms are used to

mass-produce proteins of human interest.

7.

Explain how DNA technology has helped to

produce insulin, growth hormone, and vaccines.

8.

Explain how genetically modified (GM) organisms

are transforming agriculture.

9.

Describe the risks posed by the creation and

culturing of GM organisms and the safeguards that

have been developed to minimize these risks.

(109)

10.

Describe the benefits and risks of gene therapy in

humans. Discuss the ethical issues that these

techniques present.

11.

Describe the basic steps of DNA profiling.

12.

Explain how PCR is used to amplify DNA

sequences.

13.

Explain how gel electrophoresis is used to sort

DNA and proteins.

14.

Explain how short tandem repeats are used in

DNA profiling.

You should now be able to

(110)

15.

Describe the diverse applications of DNA

profiling.

16.

Explain how restriction fragment analysis is

used to detect differences in DNA sequences.

17.

Explain why it is important to sequence the

genomes of humans and other organisms.

18.

Describe the structure and possible functions of

the noncoding sections of the human genome.

19.

Explain how the human genome was mapped.

(111)

21.

Compare the fields of genomics and proteomics.

22.

Describe the significance of genomics to the

study of evolutionary relationships and our

understanding of the special characteristics of

humans.

You should now be able to

(112)

Figure 12.UN01

Cut Bacterium

Recombinant DNA

plasmids Cut

DNA fragments

Recombinant bacteria

Bacterial clone

(113)

Figure 12.UN02

A mixture of DNA fragments

A “band” is a

collection of DNA fragments of one particular length

Longer fragments move slower

Shorter fragments move faster

DNA is attracted to  pole due to PO₄_groups

(114)

Figure 12.UN03 DNA amplified via DNA sample

treated with treated with Bacterial plasmids (a) (b) (c) DNA fragments sorted by size via

Recombinant plasmids are inserted into bacteria

(115)

Figure 12.UN03_1

DNA amplified

via

DNA sample

treated with Bacterial plasmids (a)

(b)

(116)

Figure 12.UN03_2

(c)

DNA fragments sorted by size via

Recombinant plasmids are inserted

into bacteria

are copied via (e)

(d)

Add