This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike License. Your use of this
material constitutes acceptance of that license and the conditions of use of materials on this site.
Copyright 2006, The Johns Hopkins University and Sharon Krag. All rights reserved. Use of these materials
permitted only in accordance with license rights granted. Materials provided “AS IS”; no representations or
warranties provided. User assumes all responsibility for use, and all liability related thereto, and must independently
review all materials for accuracy and efficacy. May contain materials owned by others. User is responsible for
obtaining permissions for use from third parties as needed.
Biotechnology and Genomics in Public Health
Sharon S. Krag, PhD
Johns Hopkins University
Section A
DNA Structure and Organization
DNA’s Structure: A Double-Stranded, Antiparallel Helix
Source: adapted by CTLT from Strachan, T., and Read, A. P. (1999).
Human molecular genetics, fig. 1.6 (2nd ed.). New York: Wiley-Liss.
4
A Closer Look at DNA Base Pairs
„
Two strands of DNA are non-covalently linked by hydrogen
bonds between bases on each strand. Base pair: A bonds to T;
G bonds to C
Source: adapted by CTLT from Thompson, J. N., Hellack, J. J., Braver, G., and Durica, D. S. (1997).
Chapter 3. In Primer of genetic analysis: A problems approach (p. 18). New York: Cambridge University Press.
5
How Much DNA?
How much DNA per organism?
6
Table of DNA Content in Different Organisms
DNA
Example
Number of
chromosomes
Size (bp)
Length
Plasmid
pBR322
–
4 x 103
1.3 microns
Virus
SV40
–
6 x 104
2 microns
Virus
vaccinia
–
2 x 105
100 microns
E. coli
1
4 x 106
1 mm
Yeast
S. cerevisiae
16
1.2 x 107
–
Worm
C. elegans
–
1 x 108
25 mm
Drosophilia
–
1.7 x 108
40 mm
Mouse
–
20
3 x 109
1m
Human
chromosome 21
–
5 x 107
–
Human
chromosome 1
–
3 x 108
–
Human
–
23
3 x 109
1m
Bacteria
Fly
7
Organization of DNA
How is DNA organized?
8
Gene (LDL Receptor) Organization
Source: adapted by CTLT from Gelehrter, R. D., Collins, F. S., and Ginsburg, D. (1998).
Principles of medical genetics, fig. 7.11 (2nd ed.). Baltimore: Williams and Wilkins.
9
Schema of DNA Organization in the Genome
Source: adapted by CTLT from Strachan, T., and Read, A. P. (1999).
Human molecular genetics, fig. 7.1 (2nd ed.). New York: Wiley-Liss.
10
Gene Structure
„
„
Exons
− A segment of a gene that is represented in the mature
RNA product
Introns
− Non-coding DNA which separate neighboring exons in a
gene
11
RNA Processing
Source: adapted by CTLT from Strachan, T., and Read, A. P. (1999).
Human molecular genetics, fig. 1.14 (2nd ed.). New York: Wiley-Liss.
12
Section B
Key Concepts and Approaches in Genomics
Key Concepts of Genomics
Source: CTLT
14
Making cDNA
Cells from specific
organ, tissue, or
developmental stage
(e.g., fetal brain cells)
Source: adapted by CTLT from Strachan, T., and Read, A. P. (1999).
Human molecular genetics, fig. 4.8 (2nd ed.). New York: Wiley-Liss.
15
Traditional Approach
„
Traditional approach: one gene at a time
− Gene structure
− Expression level
− Protein sequence
− Protein activity
16
Genomic Approach
„
„
Genomics—methods and approaches to study the entire
genome
Proteomics—methods and approaches to study the entire
expression complement of a system
17
Section C
Examples of Frequently Used Biotechnology Approaches
Frequently Used Biotechnologies
„
„
„
„
„
Restriction enzyme analysis
Hybridization
Sequencing
PCR
DNA arrays
19
Restriction Enzymes
These are endonucleases that cut DNA within a DNA strand. There are over
200 such enzymes, isolated from bacteria, that cut double-stranded DNA at
a specific sequence. Some of the enzymes produce blunt-ended products;
others produce “sticky-ended” products. All enzymes have a specific
sequence that they cut. Some recognize sequences of 4 bp; others as
many as 8 bp. The frequency with which a given restriction enzyme
recognition sequence occurs within a given sequence depends in part on
its length. For example, a specific 6 bp restriction site, such as the GAATTC
recognized by EcoRI, would be expected to occur in a random stretch of
DNA about once every 46 nucleotides (4,096), since there are four
possibilities (A, G, C, T) at each of the six positions.
20
Restriction Enzyme Specificity Sequences
Microorganism
Enzyme abbreviation
Sequence
HaeIII
5’ … G G C C … 3’
3’ … C C G G … 5’
Thermus aquaticus
TaqI
5’ … T C G A … 3’
3’ … A G C T … 5’
Desulfovibrio desulfuricans
DdeI
5’ … C T N A G … 3’
3’ … G A N T C … 5’
Haemophilus aegytius
EcoRV
Escherichia coli
EcoRI
Nocardia otitidis-caviarum
NotI
5’ … G
3’ … C
5’ … G
3’ … C
A T A T
T A T A
A A T T
T T A A
C … 3’
G … 5’
C … 3’
G … 5’
5’ … G C G G C C G C … 3’
3’ … C G C C G G C G … 5
Source: adapted by CTLT from Watson, J. D., Gilman, M., Witkowski, J., and Zoller, M. (1992).
Recombinant DNA, table 5.1 (2nd ed.). New York: W. H. Freeman and Company.
21
Separation Methods
Agarose gel electrophoresis is used most commonly to separate
fragments of DNA. The rate that the negatively charged DNA moves
through the agarose matrix is a function of its length, with small
fragments moving much faster than large fragments. Differently
sized fragments are separated using different concentrations of
agarose. Generally, from 0.8 to 2 percent agarose is used to separate
DNA fragments from 100 to 10,000 bp. Fragments smaller than 100
bp are separated on acrylamide gels, while fragments larger than
10,000 bp are separated by pulse-field electrophoresis.
22
Hybridization
One of the most useful techniques available for the molecular
biologist is nucleic acid (DNA or RNA) hybridization. Successful
hybridization depends on first having the molecules singlestranded. In the case of double-stranded DNA, the first step is to
denature the DNA, which means to separate it into two strands.
The phosphodiester bonds are not broken, just the hydrogen
bonds. Denaturation can be done by increasing the temperature or
treating with alkaline solution.
23
Stringency of Hybridization
Stringency of hybridization depends on the temperature, salt
concentration, and presence of organic solvents. Temperature and
organic solvents destabilize the helix, while salt stabilizes the helix.
24
Stringency of Hybridization
Source: adapted by CTLT from Gelehrter, R. D., Collins, F. S., and Ginsburg, D. (1998).
Principles of medical genetics, fig. 5.8 (2nd ed.). Baltimore: Williams and Wilkins.
25
Southern, Northern, and Western Blots
„
Explanation of Southern (separation of DNA), Northern,
(separation of RNA), and Western blots (separation of
proteins)
− These techniques, as well as dot/slot blots, utilize the
property that nucleic acid will bind tightly to
nitrocellulose filters (immobilized) and can be used in
hybridization reactions
26
Preparation of Immobilized DNA or RNA
Source: adapted by CTLT from Watson, J. D., Gilman, M., Witkowski, J., and Zoller, M. (1992).
Recombinant DNA, fig. 7.23 (2nd ed.). New York: W. H. Freeman and Company.
27
Case Study: Plasmodium falciparum DNA
1. Treat with restriction enzyme
2. Analyze on agarose gel electrophoresis
DNA probe to
gene involved in
chloroquine
resistance
–
Agarose
gel
+
28
Public Health Application
„
„
Why worry about these techniques/approaches?
Example—understanding one mechanism of drug resistance
− Chloroquine-resistant malaria parasites—why are they
resistant?
29
Drug-Resistant Parasites
„
Compare gene sequence of normal parasites and drugresistant parasites
− Changes in sequence are associated with drug resistance
30
Sequencing of DNA
Source: adapted by CTLT from Watson, J. D., Gilman, M., Witkowski, J., and Zoller, M. (1992).
Recombinant DNA (2nd ed.). New York: W. H. Freeman and Company.
31
Automated DNA Sequencing
Source: adapted by CTLT from Strachan, T., and Read, A. P. (1999). Human molecular genetics (2nd ed.).
New York: Wiley-Liss.
32
Malaria Control
„
Test a population of parasites for mutations indicating drug
resistance to inform malaria control efforts
33
PCR
„
„
PCR—the polymerase chain
reaction is a method to produce large numbers of
copies of specific DNA
sequences
There are
numerous variations
of this technique, but the
principles are delineated
below
Source: adapted by CTLT from Watson, J. D., Gilman, M.,
Witkowski, J., and Zoller, M. (1992). Recombinant DNA (2nd ed.).
New York: W. H. Freeman and Company.
34
Steps of PCR
Source: adapted by CTLT from Watson, J. D., Gilman, M., Witkowski, J., and Zoller, M. (1992).
Recombinant DNA (2nd ed.). New York: W. H. Freeman and Company.
35
Table of PCR Products
PCR Amplification of
DNA Fragment
Cycle number
Number of double-stranded
target molecules
1
0
17
32,768
2
0
18
65,536
3
2
19
131,072
4
4
20
262,144
5
8
21
524,288
6
16
22
1,048,576
7
32
23
2,097,152
8
64
24
4,194,304
9
128
25
8,388,608
10
256
26
16,777,216
11
512
27
33,544,432
12
1,024
28
67,108,864
13
2,048
29
134,217,728
14
4,096
30
268,435,456
15
8,192
31
536,870,912
16
16,384
32
1,073,741,824
Source: adapted by CTLT from Watson, J. D., Gilman, M., Witkowski, J., and Zoller, M. (1992).
Recombinant DNA, table 6.1 (2nd ed.). New York: W. H. Freeman and Company.
36
Use of PCR
„
Test a population of parasites for mutations indicating drug
resistance to inform malaria control efforts
− DNA from parasites
− PCR
− Sequencing or restriction enzyme analysis
37
DNA Microarrays
„
Hybridization using miniaturization and automation
38
Microarrays
„
„
Pre-synthesized nucleic acids
Oligonucleotides synthesized in situ
39
Microarrays
„
Microarrays are the reverse of filter hybridization techniques
we have just discussed
− Probe: set of unlabeled DNAs attached to the microarray
substrate
− Target: labeled (fluorescent) nucleic acids in solution
40
Uses of DNA Microarrays
„
„
Gene expression
Sequencing for variants (mutations or SNPs)
41