Biotechnology and Genomics in Public Health. Sharon S. Krag, PhD Johns Hopkins University

Transcription

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2 Biotechnology and Genomics in Public Health Sharon S. Krag, PhD Johns Hopkins University

3 Section A DNA Structure and Organization

4 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

5 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

6 How Much DNA? How much DNA per organism? 6

7 Table of DNA Content in Different Organisms DNA Example Number of chromosomes Size (bp) Length Plasmid pbr322 4 x microns Virus SV40 6 x microns Virus vaccinia 2 x microns Bacteria E. coli 1 4 x mm Yeast S. cerevisiae x 10 7 Worm C. elegans 1 x mm Fly Drosophilia 1.7 x mm Mouse 20 3 x m Human chromosome 21 5 x 10 7 Human chromosome 1 3 x 10 8 Human 23 3 x m 7

8 Organization of DNA How is DNA organized? 8

9 Gene (LDL Receptor) Organization Source: adapted by CTLT from Gelehrter, R. D., Collins, F. S., and Ginsburg, D. (1998). Principles of medical genetics, fig (2nd ed.). Baltimore: Williams and Wilkins. 9

10 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

11 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

12 RNA Processing Source: adapted by CTLT from Strachan, T., and Read, A. P. (1999). Human molecular genetics, fig (2nd ed.). New York: Wiley-Liss. 12

13 Section B Key Concepts and Approaches in Genomics

14 Key Concepts of Genomics Source: CTLT 14

15 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

16 Traditional Approach Traditional approach: one gene at a time Gene structure Expression level Protein sequence Protein activity 16

17 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

18 Section C Examples of Frequently Used Biotechnology Approaches

19 Frequently Used Biotechnologies Restriction enzyme analysis Hybridization Sequencing PCR DNA arrays 19

20 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 4 6 nucleotides (4,096), since there are four possibilities (A, G, C, T) at each of the six positions. 20

21 Restriction Enzyme Specificity Sequences Microorganism Enzyme abbreviation Sequence Haemophilus aegytius HaeIII 5 G G C C 3 3 C C G G 5 Thermus aquaticus Desulfovibrio desulfuricans Escherichia coli Nocardia otitidis-caviarum TaqI DdeI EcoRV EcoRI NotI 5 T C G A 3 3 A G C T 5 5 C T N A G 3 3 G A N T C 5 5 G A T A T C 3 3 C T A T A G 5 5 G A A T T C 3 3 C T T A A 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

22 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

23 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

24 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

25 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

26 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

27 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 (2nd ed.). New York: W. H. Freeman and Company. 27

28 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

29 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

30 Drug-Resistant Parasites Compare gene sequence of normal parasites and drugresistant parasites Changes in sequence are associated with drug resistance 30

31 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

32 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

33 Malaria Control Test a population of parasites for mutations indicating drug resistance to inform malaria control efforts 33

34 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

35 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

36 Table of PCR Products PCR Amplification of DNA Fragment Cycle number Number of double-stranded target molecules , , , , , , , , , , ,048, ,097, ,194, ,388, ,777, ,544, ,108, ,217, ,435, ,870, ,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

37 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

38 DNA Microarrays Hybridization using miniaturization and automation 38

39 Microarrays Pre-synthesized nucleic acids Oligonucleotides synthesized in situ 39

40 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

41 Uses of DNA Microarrays Gene expression Sequencing for variants (mutations or SNPs) 41