Chapter 20 Biotechnology. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin...

Chapter 20Chapter 20

Biotechnology

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

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 DNA

• In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule


• Methods for making recombinant DNA are central to genetic engineering (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


Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment

• To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning


I. DNA Cloning

• Use bacteria and their plasmids

– Remember: Plasmids = small circular DNA molecules that replicate separately from the bacterial chromosome

• Cloning is used for making copies of gene and producing a protein product


• Gene cloning = uses bacteria to make multiple copies of a gene

– Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell

• Reproduction of bacterial cell results in cloning of the recombinant plasmid

• Results = production of multiple copies of 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


II. Restriction Enzymes

• Bacterial restriction enzymes (protects bacteria from phages) cut DNA molecules at specific DNA sequences (restriction sites)

– Restriction enz makes many cuts, yielding restriction fragments

– Rest enz cut DNA in a staggered way, makes fragments with “sticky ends”, that bond with complementary sticky ends of other fragments

– DNA ligase = seals bonds between fragments

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


III. Cloning a Euk Gene in a Bacterial Plasmid

• cloning vector = the original plasmid that can carry foreign DNA into a host cell and replicate there


A. Producing Cells w/ Recombinant Plasmids

• Steps to clone the hummingbird β-globin gene in a bacterial plasmid:

– Isolate genomic DNA and a bacterial plasmid

– Digest both with SAME rest enz

– Fragments are mixed, DNA ligase is added to bond the fragment sticky ends


– Some recombinant plasmids now contain hummingbird DNA

– DNA mixture is added to bacteria

– Bacteria are plated on agar that selects for the bacteria with recombinant plasmids

– Results in the cloning of many hummingbird DNA fragments, including the β-globin gene

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


C3 5C CG G GA AATT T

B. Screening Clones for a Gene

• Nucleic acid probe = identifies clone by using a sequence complementary to the gene

• This process is called nucleic acid hybridization

• For example, if the desired gene is

– Then we would synthesize this probe

G5 3… …G GC C CT TTAA A


• DNA probe is used to screen a large number of clones at same time for gene of interest

• Once identified, the clone can be cultured

Fig. 20-7

ProbeDNA

Radioactivelylabeled probe

molecules

Film

Nylon membrane

Multiwell platesholding libraryclones

Location ofDNA with thecomplementarysequence

Gene ofinterest

Single-strandedDNA from cell

Nylonmembrane

TECHNIQUE

•


IV. Expressing Cloned Euk Genes

• Protein products of cloned genes can be produced in larger amounts for research

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


A. Bacterial Expression

• Scientists use an expression vector (a cloning vector that contains a highly active prokaryotic promoter) to overcome differences in promoters and other DNA control sequences


B. Eukaryotic Expression

• Using eukaryotic cells as hosts and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems

• YACs can carry more DNA than a plasmid

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


• Electroporation = applying an electrical pulse to create temp holes in plasma membranes introducing recombinant DNA

• Can inject DNA into cells using microscopically thin needles


V. Amplifying DNA in Vitro

• 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

Fig. 20-85

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

3

3

3

3

5

5

51

2

3


• DNA Cloning - Andersen

http://www.bozemanscience.com/molecule-biology


Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene

• DNA cloning allows researchers to

– Compare genes and alleles between individuals

– Locate gene expression in a body

– Determine the role of a gene in an organism

• Several techniques are used to analyze the DNA of genes


VI. Gel Electrophoresis and Southern Blotting

• Gel electrophoresis = method of rapidly analyzing and comparing genomes

– Uses gel to separate nucleic acids or proteins by size

– Electrical current is applied that causes charged molecules to move through the gel

– Molecules form “bands” by their size

Fig. 20-9

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Shortermolecules

Gel

AnodeCathode

TECHNIQUE

RESULTS

1

2

+

+

–

–


• Restriction fragment analysis = DNA fragments from rest enz digestion of DNA are sorted by gel electrophoresis

– Used for comparing two different DNA molecules

– Used to prepare pure samples of individual fragments

Fig. 20-10

Normalallele

Sickle-cellallele

Largefragment

(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

201 bp175 bp

376 bp

(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene

Normal -globin allele

Sickle-cell mutant -globin allele

DdeI

Large fragment

Large fragment

376 bp

201 bp175 bp

DdeIDdeI

DdeI DdeI DdeI DdeI

Gel Electrophoresis Virtual Lab

http://learn.genetics.utah.edu/content/labs/gel/


• Southern blotting combines gel electro with nucleic acid hybridization

– DNA fragments can be identified using labeled probes that bind to the DNA that is stuck on a “blot” of gel

Fig. 20-11a

TECHNIQUE

Nitrocellulosemembrane (blot)

Restrictionfragments

Alkalinesolution

DNA transfer (blotting)

Sponge

Gel

Heavyweight

Papertowels

Preparation of restriction fragments Gel electrophoresis

I II IIIDNA + restriction enzyme

III HeterozygoteII Sickle-cellallele

I Normal-globinallele

1 32

Fig. 20-11b

I II IIII II III

Film overblot

Probe detectionHybridization with radioactive probe

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globin allele

Probe base-pairswith fragments

Nitrocellulose blot

4 5

Radioactively labeledprobe for -globin gene


VII. DNA Sequencing

• Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths

• Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment

• The DNA sequence can be read from the resulting spectrogram

• Read from bottom to top (smallest fragment on bottom, this is 5’ end, original DNA is complimentary)

Fig. 20-12a

DNA(template strand)

TECHNIQUE

DNA polymerase

Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

dATP

dCTP

dTTP

dGTP

ddATP

ddCTP

ddTTP

ddGTP

Fig. 20-12bTECHNIQUE

RESULTS

DNA (template strand)

Shortest

Labeled strands

Longest

Shortest labeled strand

Longest labeled strand

Laser

Directionof movementof strands

Detector

Last baseof longest

labeledstrand

Last baseof shortest

labeledstrand


VIII. Analyzing Gene Expression

• Nucleic acid probes can hybridize with mRNAs transcribed from a gene, used to identify where or when a gene is transcribed in an organism

• Reverse transcriptase-polymerase chain reaction (RT-PCR)

• Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest

Fig. 20-13

TECHNIQUE

RESULTS

Gel electrophoresis

cDNAs

-globingene

PCR amplification

Embryonic stages

Primers

1 2 3 4 5 6

mRNAscDNA synthesis 1

2

3


IX. Cloning Animals

• Nuclear transplantation = nucleus of an unfertilized egg is replaced w/ nucleus of a differentiated cell

• Experiments, w/ frog embryos, have shown 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 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

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 ovary

Cells fused

Grown inculture

1

33

4

5

6

2


A. Problems with Animal Cloning

• Only a small percentage of cloned embryos have developed normally to birth

• Epigenetic changes (acetylation of histones or methylation of DNA) must be reversed in nucleus from a donor animal for genes to be expressed or repressed appropriately for development


X. Medical Applications

• Gene therapy = alteration of an afflicted individual’s genes

• Vectors are used for delivery of genes into specific types of cells, for example bone marrow, lung tissue (CFTR protein)

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


XI. Genetic Engineering in Plants

• Agricultural scientists have given a number of crop plants genes for desirable traits

• Ti plasmid = most commonly used vector for introducing new genes into plant cells

• Used to transfer herbicide resistance, inc resistance to pests, inc resistance to salinity, and improved nutritional value

Fig. 20-25

Site whererestrictionenzyme cuts

T DNA

Plant with new trait

Tiplasmid

Agrobacterium tumefaciens

DNA withthe geneof interest

RecombinantTi plasmid

TECHNIQUE

RESULTS


You should now be able to:

1. Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology

2. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid

3. Define and distinguish between genomic libraries using plasmids, phages, and cDNA

4. Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure


5. Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene

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

7. Distinguish between gene cloning, cell cloning, and organismal cloning

8. Describe how nuclear transplantation was used to produce Dolly, the first cloned sheep


9. Describe the application of DNA technology to the diagnosis of genetic disease, the development of gene therapy, vaccine production, and the development of pharmaceutical products

10.Define a SNP and explain how it may produce a RFLP

11.Explain how DNA technology is used in the forensic sciences


12.Discuss the safety and ethical questions related to recombinant DNA studies and the biotechnology industry

Chapter 20 Biotechnology. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin...

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