Chapter 17 & 19

1. Chapter 17 & 19 Genetic Engineering PLEASE READ THE CHAPTER!!!

2. Where we’re going • Basics of cloning • Polymerase chain reaction • Finding your gene in a library • DNA sequencing • Nothing controversial at this point- at least I don’t think so!

3. Definitions • Cloning: with an organism: Identical copies • Gene: poducing copies within a bacterial plasmid or phage. • Amplified PCR products (later)

4. II. Purpose‑ • Genetic Engineering allows us to isolate genes in pure form, away from thousands of other genes. This allows us to study the genes and their functions in more detail, and allows us to manipulate them to produce DNA/RNA/protein products that are of value. • It's also a lot of fun, and let's you play God, but that's secondary!

5. Overview of how:WARNING: Almost everything can now be replaced by PCR, since we have so much sequence data! • Remove the gene from one organism and place into a cloning vector in (usually) a bacteria, where it can be multiplied easily. • Vectors: plasmids and viruses. In both cases, it’s easy to grow and purify large amounts of DNA that is relatively free of other DNA. • So the trick is to stick the gene onto, or into, a plasmid or phage. • The keys restriction enzymes, ligase, and transformation or packaging procedures.

6. A. Restriction enzymes: • Almost all bacteria have within them restriction endonucleases, enzymes that recognize 4‑6 base pair sequences and break them, often producing 2‑4 bp overhangs: Fig 17-1 • 5’======GAATTC=========3’ • 3’======CTTAAG========5’ • 5’ =======G3' 5'AATTC=========3' • 3’ =======CTTAA5' + 3'G=========5'

7. Restriction enzymes • Restrict the entry of viruses • Usually palindromes- “madam,I’m adam” • ALWAYS paired w/ methylases that keep a microbe from destroying itself (makes for an interesting evolutionary puzzle)

8. Key: ANY 2 pieces of DNA cut by the same enzyme can hybridize to each other! • 5’ =======G3' 5'AATTC=========3' • 3’ =======CTTAA5' + 3'G=========5' donkey mouse However, this is not useful- it’s all going on in a microfuge tube- you’re not going to get anything; to be of value, one of these needs to be a vector; and to make it permanent, you need ligase.

9. The plasmid pUC18 offers several advantages as a vector for cloning. Because of its small size, it accepts relatively large DNA fragments for cloning; it replicates to a high copy number, and has a large number of restriction sites in the polylinker, located within a lacZ gene. Bacteria carrying pUC18 produce blue colonies when grown on media containing Xgal. DNA inserted into the polylinker site disrupts the lacZ gene; this results in white colonies and allows direct identification of colonies carrying cloned DNA inserts.

10. B/C the sticky ends are all alike, you’ll get all sorts of other products as well- if you clone ALL the red DNA into 1000’s of cells, you’d have a library Most vectors produce single cut sites for restriction enzymes!

11. .- We can also clone using YEAST- YAC’s- yeast artificial chromosomes. Fig. 17-10

12. We haven’t talked about viruses, so don’t worry much about this

13. Finding your gene- libraries and probes • IV. Cloning genes produces libraries: In normal cloning, you don’t just clone the gene you want- you produce thousands of clones, each with part of the organism’s DNA. • LIBRARY: A collection of phage or plasmid-containing cells, each with a different section of cloned DNA, and which in total contains all DNA (or mRNA) (either GENOMIC or cDNA). (With the advent of large scale sequencing, some of this is much less used)

14. Genomic and cDNA libraries • A. Genomic libraries: made with restriction enzymes, DNA, and phages or other vectors that hold large amounts of DNA. • B. cDNA libraries: made from mRNA with reverse transcriptase to turn RNA into DNA, • with the cDNA cloned into a plasmid vector. Fig 17-12

16. Then some other things happen that result in this cDNA being cloned into a vector

17. Finding the gene in question • If you can expect the gene to be expressed (properly transcribed and translated), you might be able to find it through selection or screening. • Antibiotic resistance gene • Metabolism gene- lac genes from another microbe, amino acid synthesis gene, etc.

18. V. Finding the gene in question: • To find the gene, you need a probe. Usually, the gene won’t, initially, be expressed. 17-13 • A. If not expressed: nucleotide probes: • 1. Available probes: similar genes from other species. Use at lowered stringency. • 2. oligonucleotides made from the amino acid sequence. Least degenerate 15-20 mer is used. • ala phe thr tyr trp cys asn • GC-- UU- AC- UA- UGG UG- AA- • U U U U U U • C C C C C C • A A • G G

21. Things you can do with cloned genes • Southern blots: (named after E.M. Southern): We can find a gene, and its various alleles, using Southern blots. We take chromosomal DNA and cut it w/ a restriction enzyme, run it on a gel, and blot it onto nitrocellulose paper. The blotting process transfers the DNA (now denatured) to the NC. We then hybridize with our probe- only the fragments that are complementary show up.

22. Other things you can do with cloned DNA-determine location of restriction enzyme sites(obsolete, but a good exercise in logic)- similar things are now done with PCR

23. An oldy but goody technique

26. Pa chimp Ma chimp ?? ?? ?? Think of the bands as alleles- thus only offspring A could be the offspring of mom and dad chimp

27. Other things- amounts of a gene present in cells- some cancers have amplified amounts of certain genes

28. Interpreting Southern blots: • -----*-----======*==allele A======----*--- • -----*-----======*==allele a==*===-----*-- • *******probe****** • *= restriction enzyme site. Note that the probe hybridizes to a fragment that shares at least some sequences with, but not necessarily all sequences

30. Northern Blot- this is similar, but uses RNA run on a gel, instead of DNA. Typically, it’s used to detect the presence or amount of RNA present, which will vary with tissue type or stage of development. • FYI- there is also a Western blot- protein gel, transferred, and blotted with antibodies against a specific protein. Confirming test for AIDS.

31. (The quiz on Friday) • Basics of PCR • Definitions- library, primer, probe, genomic and cDNA library & which you would use to find a gene, why cDNA libraries are needed for expression of eukaryotic genes in bacteria • Basics of Southern and northern blots.

32. The quiz • You are assigned the task of cloning Gene X from human livers. You wish to make a clone that can make the gene product. Describe how you would go about this, and how you would find the gene. • You have a similar gene from pig. • HINT- Library; cDNA; probe; reverse transcriptase; plasmid vector; transformation; mRNA

33. Isolate mRNA from human liver • Use reverse transcriptase to make cDNA • Clone the cDNA into a plasmid vector (details missing ‘cause Dr. Seelke knows, but won’t tell you) • Produce cDNA Library- lots of colonies. • Probe with the pig gene to find your gene.

34. Polymerase Chain Reaction • Another way to get your gene- and very common! • Based on a knowledge of the DNA sequence of a piece of DNA. • Allows you to design primers that, along with a thermostable DNA polymerase, let’s you make all the DNA that you need.

38. Sequencing • MANY advances in technology- highly automated • Most currently based on DNA replication of a template, and our ability to distinguish 1 nucleotide differences in the sizes of DNA.

40. Sequencing has gotten extremely cheap and easy to do- and as a result has really changed much of what we do in genetics and molecular biology.

41. Chapter 19 (highlights) • VII: Applications from CH. 19: There are lots of applications here, some of which we’ve touched on already. I will only highlight a few. This is a good chapter to use for extra credit- lots in here that we don’t touch. There’s controversy here, too.

42. FIRST • Science tells you what is- it describes reality • IT NEVER TELLS YOU WHAT OUGHT TO BE • So when a scientist tells you what you SHOULD do- s/he is being more than simply a scientist

43. My approach to controversy • 1st, and example of what isn’t my approach • Klug and Cummings, p. 443

44. Two other forms of gene therapy have not been approved, primarily because of the unresolved ethical issues surrounding them. The first is called germline therapy, whereby germ cells (the cells that give rise to the gametes…) or mature gametes are used as targets for gene transfer. In this approach, the transferred gene would be incorporated into all of the individual’s cells that are produced from the genetically altered gamete, including his or her own germ cells. This means that individuals in future generations will also be affected, without their consent. Is this procedure ethical? Do we have the right to make this decision for future generations? Thus far, the concerns have outweighed the potential benefits, and such research is prohibited.

45. Now let’s just change the subject • Another form of enhancing the racial supremacy of the Fatherland that has not been approved, primarily because of the unresolved ethical issues surrounding it. We are referring to racial cleansing whereby the inferior races, or inferior members of the Aryan race (imbeciles, mentally ill, chronic criminals) are removed from the breeding population by a large scale campaign of elimination. Is this procedure ethical? Do we have the right to make this decision for future generations? Thus far, the concerns have outweighed the potential benefits, and this policy has not been implemented.

46. Now, back to genetics… • Genetic testing: Once a gene has been identified, cloned, and sequenced, we can use this information for genetic testing. RFLP’s utilize Southern blots to detect mutant alleles in a gene.

47. (Fig. 19.9). Other technologies use probes to detect even single base change differences between normal and mutant alleles. Much of this has been extended to prenatal diagnosis, allowing the unborn child to be killed if its parent decides that she/they don’t want to deal with a “defective” child. Prenatal sex determination, resulting in the killing of (usually) a female unborn child is common in a number of countries, including China and India.

49. Genetic testing opens up fairly large “cans of worms”, some of which have already been addressed in our laws. • Screening for health insurance • Screening for jobs

50. DNA Fingerprinting • Discussed in lab