AP Biology Ch. 20

Slides for Test

DNA Cloning

• Plasmids -used to insert foreign DNA• Recombinant plasmid is inserted into a

bacteria• Reproduction in bacteria cell results in

cloning of the plasmid including foreign gene• Useful for making copies of a gene and

producing a protein product

Making recombinant DNA

• Use bacterial restriction enzymes• Cuts DNA at specific restriction sites• Cuts covalent bonds between sugar phosphate

backbone• Making restriction fragments• Most useful restriction enzymes cut DNA in a

staggered way forming “sticky ends”• DNA ligase seals the bonds between restriction

fragments.• Cloning vector is original plasmid carrying foreign

gene into the host cell.

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.

• A genomic library that is made using bacteriophages is stored as a collection of phage clones.

• A bacterial artificial chromosome (BAC)is a large plasmid that can carry a large insert with many genes.

Storing Cloned Genes in DNA Libraries

• Complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell.

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

Fig. 20-6-3

DNA innucleus

mRNAs in cytoplasm

Reversetranscriptase Poly-A tail

DNAstrand

Primer

mRNA

DegradedmRNA

Fig. 20-6-4

DNA innucleus

mRNAs in cytoplasm

Reversetranscriptase Poly-A tail

DNAstrand

Primer

mRNA

DegradedmRNA

DNA polymerase

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

• A probe can be synthesized that is complementary to the gene of interest

• For example, if the desired gene is

– Then we would synthesize this probe

G5 3… …G GC C CT TTAA A

C3 5C CG G GA AATT T

• The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest

• Once identified, the clone carrying the gene of interest 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

•

Detecting DNA sequence by hybridization with a nucleic acid probe

• Results• The location of the black spot on the photographic

film identifies the clone containing the gene of interest.

• By using probes with different nucleotide sequences, researchers can screen the collection of bacterial clones for different genes.

Expressing Cloned Eukaryotic Genes • After gene has been cloned, its protein

products can be produced in larger amounts for research.

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

Problems with expressing eukaryotic genes in bacterial host cells

• Scientists use an expression vector, a cloning vector that contains a highly active prokaryotic promoter

• This allows the bacteria to recognize the promoter and proceed to express the foreign gene.

• This allows synthesis of many eukaryotic proteins in bacteria cells.

• Another problem is the presence of introns in eukaryotic genes. • Bacteria cells do not have RNA splicing machinery. • This can be overcome by using cDNA which includes only the

exons.

Eukaryotic cloning and Expression Systems

• The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems

• YACs behave normally in mitosis and can carry more DNA than a plasmid

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

Amplifying DNA using Polymerase Chain reaction (PCR)

• PCR can produce many copies of a specific target segment of DNA

• 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

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

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

Gel Electrophoresis and Southern Blotting• Gel electrophoresis uses a gel as a molecular sieve to separate

nucleic acids by size

• A current is applied to the gel that causes the charged molecules to move

• DNA is negatively charged and it moves towards a positive pole.

• Molecules are sorted into “bands” by their size

Fig. 20-9a

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Longermolecules

Shortermolecules

Gel

AnodeCathode

TECHNIQUE

1

2

Powersource

– +

+–

Fig. 20-9b

RESULTS

• In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis

• Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

• The procedure is also 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

• A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization

• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized 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

Studying the Expression of Interacting Groups of Genes

• Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays

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

Fig. 20-15

TECHNIQUE

Isolate mRNA.

Make cDNA by reversetranscription, usingfluorescently labelednucleotides.

Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray.

Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.

Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNA fragmentsrepresentingspecific genes

DNA microarraywith 2,400human genes

DNA microarray

1

2

3

4

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

Concept 20.3: Cloning organisms may lead to production of stem

cells for research and other applications

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

Fig. 20-16

EXPERIMENT

Transversesection ofcarrot root

2-mgfragments

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

Singlecellsfree insuspensionbegan todivide.

Embryonicplant developedfrom a culturedsingle cell.

Plantlet wascultured onagar medium.Later it wasplantedin soil.

A singlesomaticcarrot celldevelopedinto a maturecarrot plant.

RESULTS

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 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

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 ovary

Cells fused

Grown inculture

1

33

4

5

6

2

• 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”

Fig. 20-19

Problems Associated 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

Concept 20.4: The practical applications of DNA technology affect our lives in many ways

• Many fields benefit from DNA technology and genetic engineering

One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

Human Gene Therapy

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

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

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

• 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 Products

• Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases

• 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 in Cell Cultures

Environmental Cleanup

• 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 Applications

• DNA technology is being used to improve agricultural productivity and food quality

Animal Husbandry• Genetic engineering of transgenic animals speeds up the

selective breeding process• Transgenic animals are made by introducing genes from

one species into the genome of another animal• Beneficial genes can be transferred between varieties or

species

Protein Production by “Pharm” Animals and Plants

• 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