1. Overview: The DNA Toolbox
Sequencing of the human genome was completed by 2007
Genetic engineering, the direct manipulation of genes for practical purposes
In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

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

3. Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment
Gene cloning produces multiple copies of gene-sized pieces of DNA.
Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids

4. DNA Cloning and Its Applications: A Preview
Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
Cloned genes are useful for making copies of a particular gene and producing a protein product

5. Figure 20.2 A preview of gene cloning and some uses of cloned genes
Cell containing gene of interest
Bacterium 1
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome 2
Plasmid put into bacterial cell
Recombinant bacterium 3
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Gene of Interest
Protein expressed by gene of interest
Copies of gene
Protein harvested
Figure 20.2 A preview of gene cloning and some uses of cloned genes 4
Basic research and various applications
Basic research on gene
Basic research on protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hor- mone treats stunted growth

6. DNA Cloning and Its Applications: A Preview
Gene cloning is useful for two basic purposes:
To make many copies of a particular gene. Isolated copies of a cloned gene may enable scientists to determine the gene’s nucleotide sequence or provide an organism with a new metabolic capability, such as pest resistance.
To create a protein product. Alternatively, a protein with medical uses, such as human growth hormone, can be harvested in large quantities from cultures of bacteria carrying the cloned gene for the protein.

7. 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

8. Restriction enzyme cuts sugar-phosphate backbones.
Fig
Restriction site
DNA 5 3 3 5 1
Restriction enzyme cuts sugar-phosphate backbones.
Sticky end
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA

9. Restriction enzyme cuts sugar-phosphate backbones.
Fig
Restriction site
DNA 5 3 3 5 1
Restriction enzyme cuts sugar-phosphate backbones.
Sticky end 2
DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA
One possible combination

10. DNA ligase is an enzyme that seals the bonds between restriction fragments

11. Restriction enzyme cuts sugar-phosphate backbones.
Fig
Restriction site
DNA 5 3 3 5 1
Restriction enzyme cuts sugar-phosphate backbones.
Sticky end 2
DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA
One possible combination 3
DNA ligase seals strands.
Recombinant DNA molecule

12. In gene cloning, the original plasmid is called a cloning vector
A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there

13. “Shotgun Approach” Fig. 20-4-4 TECHNIQUE Hummingbird cell
Bacterial cell
lacZ gene
Restriction site
Sticky ends
Gene of interest
ampR gene
Bacterial plasmid
Hummingbird DNA fragments
“Shotgun Approach”
Nonrecombinant plasmid
Thousand’s of Recombinant plasmids
Bacteria carrying plasmids
Figure 20.4 Cloning genes in bacterial plasmids
RESULTS
Colony carrying non- recombinant plasmid with intact lacZ gene
Colony carrying recombinant plasmid with disrupted lacZ gene
One of many bacterial
clones

14. 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 has been trimmed down and can carry a large DNA insert BACs are another type of vector used in DNA library construction

15. Fig. 20-5
Foreign genome cut up with restriction enzyme
Large insert with many genes
Large plasmid or
BAC clone
Recombinant phage DNA
Bacterial clones
Recombinant plasmids
Phage clones
Figure 20.5 Genomic libraries
(a) Plasmid library
(b) Phage library
(c) A library of bacterial artificial chromosome (BAC) clones

16. 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

17. 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

18. Screening a Library for Clones Carrying a Gene of Interest (Researching the library)
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

19. 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

20. 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

21. 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

22. Expressing Cloned Eukaryotic Genes (reading the book…)
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

23. Bacterial Expression Systems
Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
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

24. 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

25. 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

26. 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

27. 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

28. DNA cloning allows researchers to
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

29. Gel Electrophoresis and Southern Blotting
One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size
A current is applied that causes charged molecules to move through the gel
Molecules are sorted into “bands” by their size

30. Figure 20.9 Gel electrophoresis
TECHNIQUE
Mixture of DNA mol- ecules of different sizes
Power source –
Cathode
Anode +
Gel 1
Power source – +
Longer molecules 2
Shorter molecules
RESULTS
Figure 20.9 Gel electrophoresis

31. 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

32. 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

33. 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

34. 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

35. Fig
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 1
Preparation of restriction fragments 2
Gel electrophoresis 3
DNA transfer (blotting)
Radioactively labeled probe for -globin gene
Figure Southern blotting of DNA fragments
Probe base-pairs with fragments
I II III
I II III
Fragment from sickle-cell -globin allele
Film over blot
Fragment from normal -globin allele
Nitrocellulose blot 4
Hybridization with radioactive probe 5
Probe detection

36. 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

37. 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)

38. Disease-causing allele
Fig
DNA T
Normal allele
SNP C
Figure Single nucleotide polymorphisms (SNPs) as genetic markers for disease-causing alleles
Disease-causing allele

39. Sequencing and Analyzing Gene Expression
Dideoxyribonuclotide (dideoxy) chain termination method is an automated process that sequences the gene.

40. Studying the Expression of Single Genes
Nucleic acid probes can hybridize with mRNAs transcribed from a gene
Probes can be used to identify where or when a gene is transcribed in an organism
Changes in the expression of a gene during embryonic development can be tested using
Northern blotting
Reverse transcriptase-polymerase chain reaction
Both methods are used to compare mRNA from different developmental stages

41. Fig
In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
Figure Determining where genes are expressed by in situ hybridization analysis
50 µm

42. 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

43. 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.

44. Determining Gene Function
One way to determine function is to disable the gene and observe the consequences
Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function
Gene expression can also be silenced using RNA interference (RNAi)
Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA

45. 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
In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
The current interest in organismal cloning arises primarily from it potential to generate stem cells.

46. 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

47. 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

48. Problems Associated with Animal Cloning
Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs
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

49. 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

50. From bone marrow in this example
Fig
The aim of stem cell research is to supply cells for the repair of damaged or diseased organs
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

51. 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:
Diagnosis of Diseases
Human Gene Therapy
Pharmaceutical Products
Synthesis of Small Molecules for Use as Drugs
Protein Production in Cell Cultures
Protein Production by “Pharm” Animals and Plants

52. Human Gene Therapy
Gene therapy is the alteration of an afflicted individual’s genes

53. The practical applications continued…
Many fields benefit from DNA technology and genetic engineering:
Forensic Evidence and Genetic Profiles
Environmental Cleanup
Agricultural Applications (GMO’s)
Animal Husbandry
Genetic Engineering in Plants

54. Forensic Evidence and Genetic Profiles
Fig
(a)
Forensic Evidence and Genetic Profiles
This photo shows Earl Washington just before his release in 2001, after 17 years in prison.
short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences (use PCR to amplify)
Source of sample
STR marker 1
STR marker 2
STR marker 3
Figure STR analysis used to release an innocent man from prison
For the Discovery Video DNA Forensics, go to Animation and Video Files.
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
Kenneth Tinsley
17, 19
13, 16
12, 12
(b)
These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

55. As biotechnology continues to change, so does its use in agriculture, industry, and medicine
National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology