DNA TECHNOLOGY MAKES IT POSSIBLE TO CLONE GENES FOR BASIC RESEARCH AND COMMERCIAL APPLICATIONS

1. There once was a small black-and white-striped fish (that might even be in your aquarium) that was being studied by Dr. Mark Keating of Harvard. And Dr. Mark asked the question, “if the zebrafish can regenerate fins and eye parts, could it regenerate. . . . ?”

2. So he took this 1-inch long fish and cut away about 20% of their two chambered hearts. The incisions through the abdomen were blotted to stop bleeding, the fish were returned to the water and 8 out of 10 survived the experiment. “They sort of hang out at the bottom of the tank” it was reported. But within 10 days, the fish began to swim normally and were healthy as nonexperimental fish. After 2 months, the fish had totally regenerated their hearts, replacing all the tissue and the cells were vigorously beating with little or no scarring of the tissue. Why should we care??

3. DNA TECHNOLOGY MAKES IT POSSIBLE TO CLONE GENES FOR BASIC RESEARCH AND COMMERCIAL APPLICATIONS

4. How do you (or did you) use a bacterial plasmid to clone a gene? • Use of restriction enzymes • Mix the cut plasmid and source of the gene • -jellyfish in the pGLO lab • -but what if it was a heart muscle repair gene?

5. What occurs during the mixing of the cut plasmid and the cut DNA of interest? • 4) But how do you know that recombination has even occurred?

6. All you have at this point is, hopefully, plasmid with a new gene inserted. • 6) Introduce this modified plasmid into bacteria. • 7) How will we select for the recombinant bacteria? That is, how will you locate them? How will you know which ones have taken up the plasmid and which ones haven’t

7. Ways to distinguish the recombined bacteria: • -antibiotic resistance • -reporter gene or marker such as GFP • -if you were isolated the heart muscle tissue repair gene you could use GFP • -some kind of fluorescence • 9) Select those bacteria that have been transformed

8. But you have to have lots of the product of this gene so you must let these bacteria reproduce. • 11) So this zebrafish gene that is responsible for repairing torn heart muscle tissue might be in humans or could be implanted in human cardiac cells. • 12) How do find out if we even already have this gene but we are just not “using” it?

9. By sequencing the gene of the zebrafish and comparing its sequence to the database for our genome.

10. What has this procedure contributed to genetics? • Study gene products • Produce necessary proteins: insulin, human growth hormone, receptor proteins • Produce transgenic organisms: rice that will produce levels of vitamin A for those people / areas deficient in vit. A • Study gene regulation or gene structure • Develop a gene library • Amplify a particular gene of interest

11. Dideoxy Method of DNA Sequencing • A modified form of the normal deoxyribonucleotide triphosphate (dNTP) is used. • The modified form is called a dideoxy- because its 3’ hydroxyl is removed which prevents elongation of the DNA strand. • These dideoxy’s are also tagged with a particular molecule that when exposed to a laser will be excited and emit a different color.

12. SS DNA of interest (lots of copies) is/are isolated and mixed with: • -dNTPs • -DNA primer • -DNA polymerase • -Buffer • -fluorescent-tagged ddNTPs

13. The gene of interest is then copied but whenever a ddNTP is added to the growing strand, replication stops. • This will produce mixture of different sized fragments that are complementary to the gene of interest. • 7) The exact lengths can be used to determine the position of each base in the growing chain.

14. At this point you have fragments of ds DNA and the fluorescently tagged bases are complementary to your gene. • Heat up the DNA, separate the strands; electrophorese the different length strands (capillary electrophoresis) • As the fragments run off the gel, a laser excites the different tag on the base and emits a different color for each base. • This color and therefore base is recorded.

15. Remember, we have just determined the order of the bases in the complementary strand so the actual gene is complementary to the readout. • Dye Sequencing Animation from CSHL

16. Another method to clone a gene is by the polymerase chain reaction What were the requirements needed to perform this technique?

17. What was PCR’s contribution to genetics? • The ability to make millions of copies of a gene • Amplifies a very specific region: PCR Animation • Use in forensics • -let’s let the specific region being amplified be amplified in different amounts in various individuals. A region could be present as 1 “repeat” or 4 “repeats” or 40 “repeats.” So the region is highly variable. • -and now let’s say we have 5 to 10 different regions where various kinds of repeats could be located. • -the odds that two random individuals would share this same genetic pattern by chance is about 1 in 10 billion.

18. 4)Its use in paternity diagnosis: -same as in forensics 5) Its use in medical diagnosis: -let the Alu insert be present whenever a severe disorder of a disease occurs. This “Alu insert” would be a marker for a particular disease. Statistics would determine the probably of the development of the disease when the marker (s) is present

19. Its use in evolutionary applications • -the more closely associated DNA sequences are between organisms, the more closely related they are. • -the more specific markers, regions, genes, etc. are in common, the more associated are the organisms.

20. Figure 20.1 An overview of how bacterial plasmids are used to clone genes

21. Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA

22. Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)

23. Figure 20.4 Using a nucleic acid probe to identify a cloned gene

24. Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene

25. Figure 20.6 Genomic libraries

26. Figure 20.7 The polymerase chain reaction (PCR)

27. Figure 20.8 Gel electrophoresis of macromolecules

28. Restriction Fragment Length Patterns • RFL Patterns can be used to distinguish different alleles • Alleles are different forms of a certain gene. They can differ because of a different base sequence. This difference in base sequence then allows one form of the allele to be functional, and the other not. • Let’s say we have 2 different cloned gene samples and that the base sequence near these alleles has a restriction site or sequence. If in one of the alleles there is a different base and thus changing the restriction site, the restriction enzyme will not cut there. • This inability to cut at one of the sites will produce DNA fragments that are of different sizes / numbers. • So when the fragments from allele 1 and the fragments from allele 2 are run on a gel, different bands are produced for each of the alleles. • This shows that there is a single base difference between the two alleles of the two different DNA samples.

29. Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

30. Southern Blotting • What if we want to search for a gene of interest or a sequence of interest amongst a collection of DNA fragments? • We could “see” which fragment amongst the 100’s has our gene of interest (GOI) by attaching a radioactive probe to it. This is what a Southern Blot does, it isolates a strand of DNA by hybridization with a radioactive probe. • Purposes of Southern Blotting: • Identifies all the fragments of DNA in your sample that have the sequence of interest. • Can be used to find sequences in noncoding sections (introns) and therefore differences / similarities between organisms.

31. Figure 20.10 Restriction fragment analysis by Southern blotting Alkaline solution draws the DNA up into the nitrocellulose paper and denatures the DNA into single strands

32. RFLPs • Differences in nucleotide sequences between alleles or in the introns can create different sized fragments of DNA because these differences change the location of where a restriction enzyme can cut. • These different sized fragments are called Restriction Fragment Length Polymorphisms. • These RFLPs can serve as genetic markers telling you what your “banding pattern” looks like as compared to someone else. • Look at step 5 of the next slide

33. Figure 20.10 Restriction fragment analysis by Southern blotting Alkaline solution draws the DNA up into the nitrocellulose paper and denatures the DNA into single strands Individuals I and II have the same genetic marker.

34. Human Genome Project • It’s about: • determining the entire sequence of all 22 pairs of autosomes plus the X and Y chromosome • determining the sequence of many other species: E. coli, S. cerevisiae (yeast), Drosophila (fruit fly), Arabadopsis, mouse, C. elegans (nematode)

35. Entire Genomes Can Be Mapped • Genetic Mapping • The markers or RFLPs can be used to determine recombination frequencies which will tell you how far apart they are. • These markers could also be genes • This helps put genes “in order.”

36. Entire Genomes Can Be Mapped (cont’d) • The Ordering of DNA Fragments • You can do this by cutting up the whole chromosome with restriction enzymes and putting the fragments together. • You will use restriction enzymes that create some overlapping amongst the fragments • Chromosome walking: this helps to provide a series of markers along a chromosome • You first need to know something about a gene or marker and then make a probe, probe 1, to bind to its 3’end. • Take your DNA and cut it with two different restriction enzymes and clone copies of these fragments. • Expose your probe to Library II, the DNA of the cloned fragments cut with a different restriction enzyme.

37. Entire Genomes Can Be Mapped (cont’d) • The Ordering of DNA Fragments (cont’d) • Chromosome walking: this helps to provide a series of markers along a chromosome • Your probe 1 will bind to a fragment of the DNA from library II. • Isolate this fragment • Make a probe, probe 2, for its 3’ end. • Expose probe 2 to the DNA from library 1 and this will bind further along the DNA, hence walking down the DNA fragment. • If you keep repeating this, you move all the way down the fragment with these probes and where these probes bind serve as sequences or markers in a known order.

38. Figure 20.11 Chromosome walking

39. Figure 20.12 Sequencing of DNA by the Sanger method (Layer 1)

40. Figure 20.12 Sequencing of DNA by the Sanger method (Layer 2)

41. Figure 20.12 Sequencing of DNA by the Sanger method (Layer 3)

42. Figure 20.12 Sequencing of DNA by the Sanger method (Layer 4)

43. Figure 20.13 Alternative strategies for sequencing an entire genome

44. Table 20.1 Genome Sizes and Numbers of Genes

45. Figure 20.14a DNA microarray assay for gene expression

46. Figure 20.14b DNA microarray assay for gene expression

47. Practical Applications of DNA Technology • Diagnosis of Diseases • Knowing the sequence of a genome such as that of HIV, you can determine whether or not it is present in a blood/semen/tissue sample. • Genes for a variety of diseases have been cloned and therefore can be identified: hemophilia, cystic fibrosis, Duchenne muscular dystrophy • These genetic diseases can be detected by exposing the DNA to radioactive probes that will bind to the mutant alleles. • RFLPs can be used to identify a marker that is associated with a genetic disorder.

48. Practical Applications of DNA Technology • Human Gene Therapy • Most effective if there is a single genetic disorder. • Can we replace the gene or at least insert a good copy of it? • Insert it into the somatic cells so then then the gene product can be released into the blood stream. • Pharmaceutical Products • Human insulin • Human growth hormone • Tissue plasminogen activator: helps to dissolve blood clots and thus reduces the risk of a blood clot after a heart attack. • Protein products that block receptors (HIV) • Produce viral noninfectious protein products for vaccines

49. Figure 20.16 One type of gene therapy procedure

50. Practical Applications of DNA Technology • Forensics • The DNA sequence of each individual is unique and these variations produce different RFLPs. • Compare blood from the suspect, the victim and the crime scene to associate the suspect. • Compare blood of mother, child and possible father. • STR’s or Simple Tandem Repeats: this is where a short series of repeating bases can occur 10 to 100 times. PCR is used to selectively amplify one of these regions and will run out differently on a gel as different sized fragments distinguish you from me.