1. AP Biology Ch. 20
Slides for Test

2. 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

3. 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.

4. 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.

5. 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

6. Reverse transcriptase Poly-A tail mRNA
Fig
DNA in nucleus
mRNAs in cytoplasm
Reverse transcriptase
Poly-A tail
mRNA
DNA strand
Primer
Degraded mRNA
Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene

7. Reverse transcriptase Poly-A tail mRNA
Fig
DNA in nucleus
mRNAs in cytoplasm
Reverse transcriptase
Poly-A tail
mRNA
DNA strand
Primer
Degraded mRNA
Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene
DNA polymerase

8. Reverse transcriptase Poly-A tail mRNA
Fig
DNA in nucleus
mRNAs in cytoplasm
Reverse transcriptase
Poly-A tail
mRNA
DNA strand
Primer
Degraded mRNA
Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene
DNA polymerase
cDNA

9. 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

10. For example, if the desired gene is
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 … … 5 G G C T A A C T T A G C 3 3 C C G A T T G A A T C G 5

11. 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

12. Radioactively labeled probe molecules
Fig. 20-7
TECHNIQUE
Radioactively labeled probe molecules
Probe DNA
Gene of interest
Multiwell plates holding library
clones
Single-stranded DNA from cell
Film •
Figure 20.7 Detecting a specific DNA sequence by hybridizing with a nucleic acid probe
Nylon membrane
Nylon membrane
Location of DNA with the complementary sequence

13. 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.

14. 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.

15. 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.

16. 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

17. 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

18. molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8
TECHNIQUE 5 3
Target sequence
Genomic DNA 3 5 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
New nucleo- tides
Figure 20.8 The polymerase chain reaction (PCR)
Cycle 2 yields 4
molecules
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence

19. TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 Fig. 20-8a
Figure 20.8 The polymerase chain reaction (PCR)

20. Cycle 1 yields 2 molecules
Fig. 20-8b 1
Denaturation 5 3 3 5 2
Annealing
Cycle 1 yields 2
molecules
Primers 3
Extension
Figure 20.8 The polymerase chain reaction (PCR)
New nucleo- tides

21. Cycle 2 yields 4 molecules
Fig. 20-8c
Cycle 2 yields 4
molecules
Figure 20.8 The polymerase chain reaction (PCR)

22. molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8d
Cycle 3 yields 8
molecules; 2 molecules (in white boxes) match target sequence
Figure 20.8 The polymerase chain reaction (PCR)

23. 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

24. 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

25. Mixture of DNA mol- ecules of different sizes
Fig. 20-9a
TECHNIQUE
Power source
Mixture of DNA mol- ecules of different sizes –
Cathode
Anode +
Gel 1
Power source
Figure 20.9 Gel electrophoresis – +
Longer molecules 2
Shorter molecules

26. Fig. 20-9b
RESULTS
Figure 20.9 Gel electrophoresis

27. 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

28. Fig
Normal -globin allele
Normal allele
Sickle-cell allele
175 bp
201 bp
Large fragment
DdeI
DdeI
DdeI
DdeI
Large fragment
Sickle-cell mutant -globin allele
376 bp
201 bp 175 bp
376 bp
Large fragment
DdeI
Figure Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin gene
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

29. 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

30. Restriction fragments DNA + restriction enzyme I II III
Fig a
TECHNIQUE
Heavy weight
Restriction fragments
DNA + restriction enzyme
I II III
Nitrocellulose membrane (blot)
Gel
Sponge
I Normal -globin allele
II Sickle-cell allele
III Heterozygote
Paper towels
Alkaline solution
Figure Southern blotting of DNA fragments 1
Preparation of restriction fragments 2
Gel electrophoresis 3
DNA transfer (blotting)

31. Radioactively labeled probe for -globin gene
Fig b
Radioactively labeled probe for -globin gene
Probe base-pairs with fragments
I II III
I II III
Fragment from sickle-cell -globin allele
Film over blot
Figure Southern blotting of DNA fragments
Fragment from normal -globin allele
Nitrocellulose blot 4
Hybridization with radioactive probe 5
Probe detection

32. 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

33. Labeled cDNA molecules (single strands)
Fig
TECHNIQUE
Tissue sample 1
Isolate mRNA. 2
Make cDNA by reverse transcription, using fluorescently labeled nucleotides.
mRNA molecules
Labeled cDNA molecules (single strands) 3
Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.
DNA fragments representing specific genes
Figure DNA microarray assay of gene expression levels
DNA microarray
DNA microarray with 2,400 human genes 4
Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample.

34. 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

35. 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

36. EXPERIMENT RESULTS Transverse section of carrot root 2-mg fragments
Fig
EXPERIMENT
RESULTS
Transverse section of carrot root
2-mg fragments
Figure Can a differentiated plant cell develop into a whole plant?
Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid.
Single cells free in suspension began to divide.
Embryonic plant developed from a cultured single cell.
Plantlet was cultured on agar medium.
Later it was planted in soil.
A single somatic carrot cell developed into a mature carrot plant.

37. 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

38. EXPERIMENT RESULTS Frog embryo Frog egg cell Frog tadpole UV
Fig
Frog embryo
Frog egg cell
Frog tadpole
EXPERIMENT UV
Fully differ-
entiated
(intestinal) cell
Less differ- entiated cell
Donor nucleus trans- planted
Donor
nucleus
trans-
planted
Enucleated
egg cell
Egg with donor nucleus
activated to begin
development
RESULTS
Figure Can the nucleus from a differentiated animal cell direct development of an organism?
Most develop
into tadpoles
Most stop developing
before tadpole stage

39. 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

40. TECHNIQUE RESULTS Mammary cell donor Egg cell donor
Fig
TECHNIQUE
Mammary cell donor
Egg cell donor 1 2
Egg cell from ovary
Nucleus removed
Cultured mammary cells 3
Cells fused 3
Nucleus from mammary cell 4
Grown in culture
Early embryo
Figure Reproductive cloning of a mammal by nuclear transplantation
For the Discovery Video Cloning, go to Animation and Video Files. 5
Implanted in uterus of a third sheep
Surrogate mother 6
Embryonic development
Lamb (“Dolly”) genetically identical to mammary cell donor
RESULTS

41. 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”

42. Fig
Figure CC, the first cloned cat, and her single parent

43. 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

44. 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

45. From bone marrow in this example
Fig
Embryonic stem cells
Adult stem cells
Early human embryo at blastocyst stage (mammalian equiva- lent of blastula)
From bone marrow in this example
Cells generating all embryonic cell types
Cells generating some cell types
Cultured stem cells
Different culture conditions
Figure Working with stem cells
Different types of differentiated cells
Liver cells
Nerve cells
Blood cells

46. The aim of stem cell research is to supply cells for the repair of damaged or diseased organs

47. Many fields benefit from DNA technology and genetic engineering
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

48. 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

49. Insert RNA version of normal allele into retrovirus.
Fig
Cloned gene 1
Insert RNA version of normal allele into retrovirus.
Viral RNA 2
Let retrovirus infect bone marrow cells that have been removed from the patient and cultured.
Retrovirus capsid 3
Viral DNA carrying the normal allele inserts into chromosome.
Bone marrow cell from patient
Figure Gene therapy using a retroviral vector
Bone marrow 4
Inject engineered cells into patient.

50. Pharmaceutical Products
Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases

51. Protein Production in Cell Cultures
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

52. 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

53. Agricultural Applications
DNA technology is being used to improve agricultural productivity and food quality

54. Animal Husbandry Protein Production by “Pharm” Animals and Plants
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