1. Genetic Technology

2. Learning Objectives By the end of this class you should understand:
The concept and mechanics of cloning
The purpose and methodology of DNA restriction
Methods relating to keeping libraries of genes
The purpose and mechanics of PCR
The purpose and mechanics of a Southern blot and microarray analysis

3. Genetic Technology
So far material has focused on the existing system of DNA
Have to know the system before you can propose manipulating it!

4. Cloning
Cloning has been the subject of fiction long before it became fact
Cloned cells are genetically identical
Cloned individuals do NOT share thoughts or memories since those are not genetic

5. Cloning Methods Embryo Splitting Nuclear Fusion
Essentially intentional creation of identical twins/triplets
Nuclear Fusion
Insertion of a nucleus into an enucleated egg and then implanting the egg into a surrogate mother

6. Enucleated Egg
Removal of a nucleus from an egg cell creates an enucleated egg
This is useful because it still contains all the cytoplasmic factors needed to develop into an embryo
Fusion was originally performed with an electrical shock, now performed with a microneedle

7. Cloning Process

8. Why Clone?
An animal that has a particular advantage can be bred with other animals but regression to the mean will likely occur
Cloning produces entire herds of cattle or sheep that are the most effective at producing milk/wool/etc
Downside: clones are all vulnerable to the same diseases!

9. Gene Cloning
Another major technique is to clone a section of DNA rather than an entire organism
Known as recombinant DNA technology
Re-combining (mixing- and-matching) DNA
Required to place individual genes into living things

10. Key Recombinant DNA Tool
The most useful tool in cloning genes is called a restriction enzyme
Restriction enzymes cut DNA in a distinctive pattern that leaves “sticky ends”
Only cuts very precise sequences of DNA

11. Restriction Enzymes
Originally discovered in bacteria as virus protection
Useful now to insert pieces of DNA into other pieces of DNA

12. Restriction Enzyme Activity
If two DNA strands with the same restriction sequences are mixed and restricted they will then pair indiscriminately
To reseal the DNA, the enzyme DNA ligase must be used
Same enzyme that attaches Okazaki fragments!

13. Bacterial Plasmids
Bacteria have a bacterial chromosome with all their DNA, but they may also carry small “bonus gene” carriers called bacterial plasmids
May carry genes for toxins or antibiotic resistance
May be traded to other bacteria
May be injected into bacteria

14. Bacterial Plasmid Use
Different plasmids have restriction enzyme sites, and may have genes inserted into them using restriction enzymes
In this manner, bacteria may be induced to express any gene!

15. pBR322 Standard plasmid for use in gene cloning
Has gene inserted via restriction enzymes
Injected into bacteria
Becomes a vector for cloned gene
Sometimes include an antibiotic resistance gene in vector to weed out other bacteria

16. Recombinant Plasmids

17. Storage of Cloned Genes
Genes can be stored in a genomic library of bacterial colonies
Genes can be located using a probe made of DNA which matches only a certain gene
If bacterial colonies are mixed together, a radioactive probe can identify the desired colony

18. DNA Probe

19. Storage of Chromosomes
Larger sequences of DNA may be stored also
Plasmid can only hold one gene
Yeast Artificial Chromosome is useful for storing large sections of DNA
Up to 1 million bases (several hundred genes)

20. Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a gene cloning technique invented in the 1980s
Produces billions of copies of a gene in hours
Essentially, DNA strands are separated and mixed with free DNA bases and Taq DNA Polymerase
DNA Polymerase from a hot springs bacterium so it can handle the heat

21. PCR Diagram

22. PCR Part 2

23. PCR Part 3

24. PCR Process Demonstration:
e.com/watch?v= _YgXcJ4n-kQ

25. PCR Applications
If a single hair is taken from a crime scene, is there enough DNA to analyze?
If not, use PCR!
DNA can be cut with restriction enzymes into different lengths and run in a Southern Blot
Each bar is a different length of DNA
Smaller = faster

26. Southern Blot Identity
If two identical strands of DNA are restricted by the same enzyme they will produce the same pattern
Smaller pieces move faster than larger ones
This is how DNA fingerprinting works!

27. Southern Blot Results
DNA is bound with radioactive probes or luminescent DNA stain
Result shows different patterns because different DNA has different restriction sites

28. Microarray Analysis
Microarrays are used to study gene expression rather than gene presence
Southern blot will show same results for all cells from a particular human
Even cancerous cells will look similar

29. Microarray Procedure
Each well contains complementary DNA (cDNA) that matches a mRNA for a gene
When a tissue sample is lysed, its mRNAs will bind to the cDNA of each well
Different cDNAs are labeled different colors

30. Microarray Analysis
When two different tissues are put in simultaneously, each is associated with a particular dye
For example, normal cell expression is green and cancer cell expression is red
This shows which genes are no longer expressed and which are newly expressed in cancer cells

31. See you tomorrow!