1. DNA Technology and Genomics
Chapter 12
DNA Technology and Genomics

2. DNA and Crime Scene Investigations
Many violent crimes go unsolved
lack of enough evidence
If biological fluids are left at a crime scene
DNA can be isolated from them

3. DNA fingerprinting: set of laboratory procedures
determines w/near certainty whether 2 samples of DNA are from same individual
powerful tool for CSIs
Investigator at one
of the crime scenes
(above), Narborough,
England (left)

4. BACTERIAL PLASMIDS AND GENE CLONING
12.1 Plasmids are used to customize bacteria: An overview
Gene cloning is 1 application
DNA technology
Methods for studying & manipulating genetic material

5. And insert those plasmids into bacteria
Researchers can insert desired genes into plasmids, creating recombinant DNA
And insert those plasmids into bacteria
Figure 12.1

6. Bacterium Bacterial Plasmid chromosome Figure 12.1 Plasmid
isolated
Cell containing gene
of interest 2
DNA
isolated
Bacterial
chromosome 3
Gene inserted
into plasmid
Plasmid
DNA
Recombinant DNA
(plasmid)
Gene of
interest 4
Plasmid put into
bacterial cell
Recombinant
bacterium
Gene for pest
Resistance inserted into plants 5
Cell multiplies with
gene of interest
Copies of gene
Copies of protein
Clone of cells
Protein used to make snow form at higher temperature
Figure 12.1
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to dissolve blood clots in heart attack therapy

7. If the recombinant bacteria multiply into a clone
The foreign genes are also copied

8. 12.2 Enzymes are used to “cut and paste” DNA
The tools used to make recombinant DNA are
Restriction enzymes, which cut DNA at specific sequences
DNA ligase, which “pastes” DNA fragments together

9. Restriction enzyme recognition sequence
Creating recombinant DNA using REs & DNA ligase
DNA
G A A T T C
C T T A A G 1
Restriction enzyme recognition sequence
Restriction enzyme cuts the DNA into fragments G
A AT TC
C T T A A 2 G
Sticky end
A AT TC G
Addition of a DNA fragment from another source G 3
C T T A A
Two (or more) fragments stick together by base-pairing
G A AT T C
C T TA A G
G A AT T C
C T TA A G 4
DNA ligase
pastes the strand
Recombinant DNA molecule 5
Figure 12.2

10. 12.3 Genes can be cloned in recombinant plasmids: A closer look
12.3 Genes can be cloned in recombinant plasmids: A closer look
Bacteria take the recombinant plasmids from their surroundings
reproduce
cloning the plasmids and the genes they carry

11. Cloning a gene in a bacterial plasmid1
Isolate DNA from two sources
E.coli
Human cell
DNA 2
Cut both DNAs with the same restriction enzyme
Plasmid
Gene V
Sticky ends 3
Mix the DNAs; they join by base-pairing 4
Add DNA ligase to bond the DNA covalently
Recombinant DNA plasmid
Gene V 5
Put plasmid into bacterium by transformation
Recombinant bacterium 6
Clone the bacterium
Figure 12.3
Bacterial clone carrying many copies of the human gene

12. CONNECTION
12.6 Recombinant cells and organisms can mass-produce gene products
Applications of gene cloning include
medical and other uses
Table 12.6

13. Different organisms, including bacteria, yeast, and mammals
Different organisms, including bacteria, yeast, and mammals
Can be used for this purpose
Figure 12.6

14. 12.7 DNA technology is changing the pharmaceutical industry
CONNECTION
12.7 DNA technology is changing the pharmaceutical industry
widely used to produce medicines and to diagnose diseases

15. Therapeutic hormones
In 1982, humulin, human insulin produced by bacteria
1st recombinant drug approved by FDA
Figure 12.7A

16. Diagnosis and Treatment of Disease
Diagnosis and Treatment of Disease
DNA technology
Is being used increasingly in disease diagnosis

17. Vaccines DNA technology
Vaccines
DNA technology
Is also helping medical researchers develop vaccines
Figure 12.7B

18. pGLO LAB

19. RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING
12.10 Gel electrophoresis sorts DNA molecules by size + –
Power
source
Gel
Mixture of DNA
molecules of
different sizes
Longer
molecules
Shorter
Completed gel
Figure 12.10

20. 12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

21. How Restriction Fragments Reflect DNA Sequence
How Restriction Fragments Reflect DNA Sequence
Restriction fragment length polymorphisms (RFLPs)
Reflect differences in the sequences of DNA samples
Crime scene
Suspect w x y z
Cut
DNA from chromosomes C G A T
Figure 12.11A

22. After digestion by restriction enzymes fragments are run through a gel
After digestion by restriction enzymes
fragments are run through a gel – +
Longer
fragments
Shorter x w y z 1 2
Figure 12.11B

23. Using DNA Probes to Detect Harmful Alleles
Using DNA Probes to Detect Harmful Alleles
Radioactive probes
Can reveal DNA bands of interest on a gel

24. Detecting a harmful allele using restriction fragment analysis
Detecting a harmful allele using restriction fragment analysis 1 2 3 4 5
Restriction fragment
preparation
Gel electrophoresis
Blotting
Radioactive probe
Detection of radioactivity
(autoradiography) I II
III
Restriction
fragments
Filter paper
Probe
Radioactive, single-
stranded DNA (probe)
Film
Figure 12.11C

25. CONNECTION
12.12 DNA technology is used in courts of law

26. DNA fingerprinting can help solve crimes
DNA fingerprinting can help solve crimes
Figure 12.12B
Defendant’s
blood
Blood from
defendant’s clothes
Victim’s
Figure 12.12A

27. CONNECTION
12.13 Gene therapy may someday help treat a variety of diseases
Gene therapy
Is the alteration of an afflicted individual’s genes

28. Gene Therapy Cloned gene (normal allele) 1 Insert normal gene
into virus
Retrovirus
Viral nucleic
acid 2
Infect bone marrow
cell with virus 3
Viral DNA inserts
into chromosome
Bone marrow
cell from patient 4
Inject cells
into patient
Bone
marrow
Figure 12.13

29. Gene therapy
May one day be used to treat both genetic diseases and nongenetic disorders
Unfortunately, progress is slow

30. 12.14 The PCR method is used to amplify DNA sequences
12.14 The PCR method is used to amplify DNA sequences
The polymerase chain reaction (PCR)
Can be used to clone a small sample of DNA quickly, producing enough copies for analysis 1 2 4 8
Initial
DNA
segment
Number of DNA molecules
Figure 12.14

31. GENOMICS CONNECTION
12.15 The Human Genome Project is an ambitious application of DNA technology
The Human Genome Project, begun in 1990 and now largely completed
Genetic and physical mapping of chromosomes, followed by DNA sequencing
Figure 12.15

32. The data are providing insight into
Development, evolution, and many diseases

33. 12.16 Most of the human genome does not consist of genes
12.16 Most of the human genome does not consist of genes
The haploid human genome contains about 25,000 genes
And a huge amount of noncoding DNA

34. Much of the noncoding DNA consists of repetitive nucleotide sequences
And transposons that can move about within the genome

35. 12.17 The science of genomics compares whole genomes
CONNECTION
12.17 The science of genomics compares whole genomes
The sequencing of many prokaryotic and eukaryotic genomes

36. Besides being interesting themselves...
Nonhuman genomes can be compared with the human genome
Table 12.17

37. Proteomics
Is the study of the full sets of proteins produced by organisms

38. GENETICALLY MODIFIED ORGANISMS: CONNECTION
12.18 Genetically modified organisms are transforming agriculture

39. gene within plant chromosome
Recombinant DNA technology
Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms
Agrobacterium tumefaciens
DNA containing
gene for desired trait
Plant cell Ti
plasmid 1
Insertion of gene
into plasmid using
restriction enzyme
and DNA ligase 2
Introduction
into plant
cells in
culture 3
Regeneration
of plant
Recombinant
Ti plasmid
T DNA carrying new
gene within plant chromosome
T DNA
Plant with new trait
Restriction
site
Figure 12.18A

40. have had genes from other organisms inserted into their genomes
Transgenic organisms
have had genes from other organisms inserted into their genomes
Figure 12.18B

41. A number of important crops and plants
Are genetically modified

42. 12.19 Could GM organisms harm human health or the environment?
CONNECTION
12.19 Could GM organisms harm human health or the environment?
Development of GM organisms
Requires significant safety measures
Figure 12.19A

43. Genetic engineering involves risks
Genetic engineering involves risks
Such as ecological damage from GM crops
Figure 12.19B

44. CONNECTION
12.20 Genomics researcher Eric Lander discusses the Human Genome Project
Genomics pioneer Eric Lander
Points out that much remains to be learned from the Human Genome Project
Figure 12.20

46. 12.17 The science of genomics compares whole genomes
12.17 The science of genomics compares whole genomes
The sequencing of many prokaryotic and eukaryotic genomes
Has produced data for genomics, the study of whole genomes

47. 12.4 Cloned genes can be stored in genomic libraries
12.4 Cloned genes can be stored in genomic libraries
Genomic libraries, sets of DNA fragments containing all of an organism’s genes
Can be constructed and stored in cloned bacterial plasmids or phages
Recombinant plasmid
Genome cut up with restriction enzyme
Recombinant phage DNA or
Bacterial clone
Phage clone
Phage library
Plasmid library
Figure 12.4

48. 12.5 Reverse transcriptase helps make genes for cloning
12.5 Reverse transcriptase helps make genes for cloning
Reverse transcriptase can be used to make smaller, complementary DNA (cDNA) libraries
Containing only the genes that are transcribed by a particular type of cell
Cell nucleus
DNA of eukaryotic gene
Exon
Intron
Exon
Intron
Exon 1
Transcription
RNA transcript 2
RNA splicing (removes introns)
mRNA 3
Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand
Test tube
Reverse transcriptase
cDNA strand 4
Breakdown of RNA 5
Synthesis of second DNA strand
cDNA of gene (no introns)
Figure 12.5

49. RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING
12.8 Nucleic acid probes identify clones carrying specific genes
DNA technology methods
Can be used to identify specific pieces of DNA

50. Can tag a desired gene in a library
A nucleic acid probe
Is a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA
Can tag a desired gene in a library
Radioactive
probe (DNA)
Single-stranded
DNA
Mix with single-
stranded DNA from
various bacterial
(or phage) clones
Base pairing
indicates the
gene of interest
A T C C G A
A T G C G C T T A T C G
A G C C T T A T G C A T
A T C C G A
A G G T A G G C T A A
Figure 12.8

51. 12.9 DNA microarrays test for the expression of many genes at once
CONNECTION
12.9 DNA microarrays test for the expression of many genes at once
DNA microarray assays
Can reveal patterns of gene expression in different kinds of cells

52. DNA microarray Figure 12.9 DNA microarray Each well contains DNA
DNA microarray
DNA microarray
Each well contains DNA
from a particular gene
Actual size
(6,400 genes) 1
mRNA
isolated 4
Unbound
cDNA rinsed
away
Reverse transcriptase
and fluorescent DNA
nucleotides
Fluorescent
spot
Nonfluorescent
spot 3
cDNA applied
to wells 2
cDNA made
from mRNA
cDNA
DNA of an
expressed gene
DNA of an
unexpressed gene
Figure 12.9