28.13 AIDS

1. 28.13AIDS

2. AIDS • Acquired Immune Deficiency Syndrome • More than 22 million people have died from AIDS since disease discovered in 1980s. • Now fourth leading cause of death worldwide and leading cause of death in Africa (World Health Organization).

3. HIV • Virus responsible for AIDS in people is Human Immunodeficiency Virus (HIV). • Several strains of HIV designated HIV-1, HIV-2, etc. • HIV is a retrovirus. Genetic material is RNA, not DNA.

4. HIV • HIV inserts its own RNA and an enzyme (reverse transcriptase) in T4 lymphocyte cell of host. • Reverse transcriptase catalyzes the formation of DNA complementary to the HIV RNA. • HIV reproduces and eventually infects other T4 lympocytes. • Ability of T4 cells to reproduce decreases, interfering with bodies ability to fight infection.

5. O O H3C O O N NH NH HOCH2 HOCH2 N N N O H H H H N3 AZT ddI AIDS Drugs • AZT and ddI are two drugs used against AIDS that delay onset of symptoms.

6. AIDS Drugs • Protease inhibitors are used in conjunction with other AIDS drugs. • Several HIV proteins are present in the same polypeptide chain and must be separated from each other in order to act. • Protease inhibitors prevent formation of HIV proteins by preventing hydrolysis of polypeptide that incorporates them.

7. 28.14DNA Sequencing

8. DNA Sequencing • Restriction enzymes cleave the polynucleotide to smaller fragments. • These smaller fragments (100-200 base pairs) are sequenced. • The two strands are separated.

9. OH OH OH base HO O POCH2 O P P O O O O HO H DNA Sequencing • Single stranded DNA divided in four portions. • Each tube contains adenosine, thymidine, guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs.

10. OH OH OH base HO O POCH2 O P P O O O O H H DNA Sequencing • The first tube also contains the 2,'3'-dideoxy analog of adenosine triphosphate (ddATP); the second tube the 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), the third contains ddGTP, and the fourth ddCTP.

11. DNA Sequencing • Each tube also contains a "primer", a short section of the complementary DNA strand, labeled with radioactive phosphorus (32P). • DNA synthesis takes place, producing a complementary strand of the DNA strand used as a template. • DNA synthesis stops when a dideoxynucleotide is incorporated into the growing chain.

12. DNA Sequencing • The contents of each tube are separated by electrophoresis and analyzed by autoradiography. • There are four lanes on the electrophoresis gel. • Each DNA fragment will be one nucleotide longer than the previous one.

13. ddA ddT ddG ddC T TG TGA TGAC TGACA TGACAT TGACATA TGACATAC TGACATACG TGACATACGT Sequence of fragment Figure 28.13

14. ddA ddT ddG ddC T A TG AC TGA ACT TGAC ACTG TGACA ACTGT TGACAT ACTGTA TGACATA ACTGTAT TGACATAC ACTGTATG TGACATACG ACTGTATGC TGACATACGT ACTGTATGCA Sequence of fragment Sequence of original DNA Figure 28.13

15. 28.15The Human Genome Project

16. Human Genome Project • In 1988 National Research Council (NRC) recommended that the U.S. undertake the mapping and sequencing of the human genome. • International Human Genome Sequencing Consortium (led by U.S. NIH) and Celera Genomics undertook project. Originally competitors, they agreed to coordinate efforts and published draft sequences in 2001.

17. 28.16DNA Profilingand thePolymerase Chain Reaction

18. DNA Profiling • DNA sequencing involves determining the nucleotide sequence in DNA. • The nucleotide sequence in regions of DNA that code for proteins varies little from one individual to another, because the proteins are the same. • Most of the nucleotides in DNA are in "noncoding" regions and vary significantly among individuals. • Enzymatic cleavage of DNA give a mixture of polynucleotides that can be separated by electrophoresis to give a "profile" characteristic of a single individual.

19. PCR • When a sample of DNA is too small to be sequenced or profiled, the polymerase chain reaction (PCR) is used to make copies ("amplify") portions of it. • PCR amplifies DNA by repetitive cycles of the following steps. • 1. Denaturation 2. Annealing ("priming") 3. Synthesis ("extension" or "elongation")

20. Figure 28.14: (PCR) (a) Consider double-stranded DNA containinga polynucleotide sequence (the target region)that you wish to amplify. Target region (b) Heating the DNA to about 95°C causes thestrands to separate. This is the denaturation step.

21. Figure 28.14: (PCR) (c) Cooling the sample to ~60°C causes oneprimer oligonucleotide to bind to one strand andthe other primer to the other strand. This is theannealing step. (b) Heating the DNA to about 95°C causes thestrands to separate. This is the denaturation step.

22. Figure 28.14: (PCR) (c) Cooling the sample to ~60°C causes oneprimer oligonucleotide to bind to one strand andthe other primer to the other strand. This is theannealing step. (d) In the presence of four DNA nucleotides andthe enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesisstep and is carried out at 72°C.

23. Figure 28.14: (PCR) This completes one cycle of PCR. (d) In the presence of four DNA nucleotides andthe enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesisstep and is carried out at 72°C.

24. Figure 28.14: (PCR) This completes one cycle of PCR. (e) The next cycle begins with the denaturationof the two DNA molecules shown. Both arethen primed as before.

25. Figure 28.14: (PCR) (f) Elongation of the primed fragments completesthe second PCR cycle. (e) The next cycle begins with the denaturationof the two DNA molecules shown. Both arethen primed as before.

26. (g) Among the 8 DNAs formed in the secondcycle are two having the structure shown. Figure 28.14: (PCR) (f) Elongation of the primed fragments completesthe second PCR cycle.

27. (g) Among the 8 DNAs formed in the secondcycle are two having the structure shown. Figure 28.14: (PCR) The two contain only the target region andand are the ones that increase disproportionately in subsequent cycles.

28. Table 28.4 Cycle Total DNAs Contain only target 0 (start) 1 0 1 2 0 2 4 0 3 8 2 4 16 8 5 32 22 10 1,024 1,00420 1,048,566 1,048,526 30 1,073,741,824 1,073,741,764