1. Chapter 20: Biotechnology

2. 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

3. Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes
DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products

4. DNA Cloning
Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids
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. Gene cloning involves using 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 in the bacterial cell results in cloning of the plasmid including the foreign DNA
This results in the production of multiple copies of a single gene

6. 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

7. Cell containing gene of interest Bacterium
Fig. 20-2a
Cell containing gene of interest
Bacterium 1
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome 2 2
Plasmid put into bacterial cell
Figure 20.2 A preview of gene cloning and some uses of cloned genes
Recombinant bacterium

8. Recombinant bacterium
Fig. 20-2b
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 4
Basic research and various applications
Basic research on gene
Basic research on protein
Figure 20.2 A preview of gene cloning and some uses of cloned genes
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

9. 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

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
Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA

12. 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

13. 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

14. Cloning a Eukaryotic Gene in a Bacterial Plasmid
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

15. Producing Clones of Cells Carrying Recombinant Plasmids
Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid:
The hummingbird genomic DNA and a bacterial plasmid are isolated
Both are digested with the same restriction enzyme
The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends

16. Some recombinant plasmids now contain hummingbird DNA
The DNA mixture is added to bacteria that have been genetically engineered to accept it
The bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids
This results in the cloning of many hummingbird DNA fragments, including the β-globin gene

17. Fig
TECHNIQUE
Hummingbird cell
Bacterial cell
lacZ gene
Restriction site
Sticky ends
Gene of interest
ampR gene
Bacterial plasmid
Hummingbird DNA fragments
Figure 20.4 Cloning genes in bacterial plasmids

18. Fig
TECHNIQUE
Hummingbird cell
Bacterial cell
lacZ gene
Restriction site
Sticky ends
Gene of interest
ampR gene
Bacterial plasmid
Hummingbird DNA fragments
Nonrecombinant plasmid
Recombinant plasmids
Figure 20.4 Cloning genes in bacterial plasmids

19. Fig
TECHNIQUE
Hummingbird cell
Bacterial cell
lacZ gene
Restriction site
Sticky ends
Gene of interest
ampR gene
Bacterial plasmid
Hummingbird DNA fragments
Nonrecombinant plasmid
Recombinant plasmids
Bacteria carrying plasmids
Figure 20.4 Cloning genes in bacterial plasmids

20. Fig
TECHNIQUE
Hummingbird cell
Bacterial cell
lacZ gene
Restriction site
Sticky ends
Gene of interest
ampR gene
Bacterial plasmid
Hummingbird DNA fragments
Nonrecombinant plasmid
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

21. 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

22. 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

23. 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

24. Then we would synthesize this probe
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

25. 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

26. 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

27. Expressing Cloned Eukaryotic Genes
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

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive
Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest
The products are run on a gel and the mRNA of interest identified

35. TECHNIQUE 1 cDNA synthesis mRNAs cDNAs Primers 2 -globin gene 3
Fig
TECHNIQUE 1
cDNA synthesis
mRNAs
cDNAs
Primers 2
PCR amplification
-globin gene 3
Gel electrophoresis
Figure RT-PCR analysis of expression of single genes
Embryonic stages
RESULTS

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

37. 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, can become any cell in body

38. 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.

39. 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
The older the donor nucleus, the lower the percentage of normally developing tadpoles

40. 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

41. 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

42. 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

43. 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”

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

45. 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

46. 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

47. 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.

48. Fig
(a)
This photo shows Earl Washington just before his release in 2001, after 17 years in prison.
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.

49. Agrobacterium tumefaciens
Fig
TECHNIQUE
Agrobacterium tumefaciens
Ti plasmid
Site where restriction enzyme cuts
T DNA
RESULTS
DNA with the gene of interest
Figure Using the Ti plasmid to produce transgenic plants
For the Cell Biology Video Pronuclear Injection, go to Animation and Video Files.
For the Discovery Video Transgenics, go to Animation and Video Files.
Recombinant Ti plasmid
Plant with new trait

50. The Sanger Method (DNA Sequencing)
Nobel prize in 1980 for sequencing DNA
Uses dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators ( 4 exist A, T, C, G)
DNA separated, primers added, DNA polymerase adds nucelotides
ddNTPs added to terminate building of DNA
Run a gel to determine fragment length, DNA sequencing

51. Human Genome Project Sequencing of the human genome
Human Genome Project began in 1990, the sequencing was largely completed by 2003
The project had three stages:
Genetic (or linkage) mapping
Restriction Fragment Length Polymorphisms (RFLPs) - differences in single nucleotides that change the fragment length, enzyme cuts in different place
Known as a genetic marker (identifiable DNA sequence)
Physical mapping- finding the distances between genetic markers
DNA sequencing- determine complete nucleotide sequencing

52. Non Coding DNA
Pseudogenes are former genes that have accumulated mutations and are nonfunctional
Repetitive DNA is present in multiple copies in the genome
Where might you find repetitive DNA?

53. Transposable Elements
The first evidence for wandering DNA segments came from geneticist Barbara McClintock’s breeding experiments with Indian corn

54. Transposable Elements
75% of repetitive DNA
Transposable elements move from one site to another in a cell’s DNA; they are present in both prokaryotes and eukaryotes
Transposons: move within a genome by means of a DNA intermediate (enzyme)
Who cares?: Can lead to small variations within species
Changes the size of RFLPs (genetic markers)
*What would happen if a transposon interuppted a gene?

55. Transposon is copied New copy of retrotransposon Reverse transcriptase
Fig. 21-9
New copy of
transposon
Transposon
DNA of
genome
Transposon
is copied
Insertion
Mobile transposon
(a) Transposon movement (“copy-and-paste” mechanism)
New copy of
retrotransposon
Retrotransposon
Figure 21.9 Movement of eukaryotic transposable elements
RNA
Insertion
Reverse
transcriptase
(b) Retrotransposon movement

56. Tandem Repeats
Short Tandem Repeat: A series of repeating units of 2 to 5 nucleotides
The repeat number for STRs can vary among sites (within a genome) or individuals
STRs are common in centromeres and telomeres, where it probably plays structural roles in the chromosome

57. Mutation leads to Variation/Evolution
The basis of change at the genomic level is mutation, which underlies much of genome evolution
Causes of mutations?
Insertions, Deletions, Inversions, Transposons, accidents in meiosis, errors or duplications when exon shuffling
*But we show many similarities between species!

58. To get you thinking…
Insertion of transposable elements within a protein-coding sequence may block protein production
Insertion of transposable elements within a regulatory sequence may increase or decrease protein production

59. Humans Vs. Chimps
Human and chimpanzee genomes differ by 1.2%, at single base-pairs, and by 2.7% because of insertions and deletions
Several genes are evolving faster in humans than chimpanzees
These include genes involved in defense against malaria and tuberculosis, regulation of brain size, and genes that code for transcription factors
Why is this significant?

60. FOXP2 Gene
Humans and chimpanzees differ in the expression of the FOXP2 gene whose product turns on genes involved in vocalization
Differences in the FOXP2 gene may explain why humans but not chimpanzees communicate by speech

61. Comparing Genomes Within a Species
As a species, humans have only been around about 200,000 years and have low within-species genetic variation
Variation within humans is due to single nucleotide polymorphisms, inversions, deletions, and duplications, transposons changing RFLP size
These variations are useful for studying human evolution and human health

62. Conservation of Developmental Genes
Molecular analysis of the homeotic genes in Drosophila has shown that they all include a sequence called a homeobox ( sequence of 180 nucleotides)
An identical or very similar nucleotide sequence found in homeotic genes of both vertebrates and invertebrates
Responsible for laying out body plan (segments)
A gene that controls all the genes involved in development

63. Adult fruit fly Fruit fly embryo (10 hours) Fly chromosome Mouse
Fig
Adult
fruit fly
Fruit fly embryo
(10 hours)
Fly
chromosome
Mouse
chromosomes
Figure Conservation of homeotic genes in a fruit fly and a mouse
Mouse embryo
(12 days)
Adult mouse