2. Biology 417 Week 1, Lecture #2 With input from: Yung Huang, Luis Sanchez, Lee Lin, Leticia Argueta, Kay Nguyen PGM 2000 Revised 2012 - SBS

3. Schedule for today Quiz Go over quiz Announcements Lecture

4. Announcements Homework 1 due Friday. Before next Monday, please carefully read the description and assessment sheets for the term assignment. Teams and topics will be announced Monday.

5. Outline, Lecture 2, Week 1 Some basic definitions Why get enough of YFG How to get enough of YFG In vivo In vitro Restriction/methylation systems Basic research discovers a way to make recombinant DNA work more predictable.

6. Definitions Gene manipulation (also known as genetic engineering) The use of molecular and informatic (computer) techniques to produce DNA molecules containing new genes or new combinations of genes produce specialized vectors or vehicles for carrying recombinant DNA in cells Examples of vectors: plasmids, cosmids, lambda DNA, filamentous phage DNA, retrovirus DNA, lentivirus DNA, BAC, YAC, P1 study genes and gene expression

7. http://pmj.bmj.com/content/79/931/249/F1.large.jpg

8. Figure Biotechnology patents issued by US Patent and Trademark Office 1996?2000 Source: USPTO's patent full text and image database. John R La Montagne Biotechnology and research: promise and problems The Lancet Volume 358, Issue 9294 2001 1723 - 1724 http://dx.doi.org/10.1016/S0140-6736(01)06717-4

9. http://www.nature.com/nbt/journal/v25/n12/box/nbt1207-1341_BX4.html

10. Definitions One branch of Biotechnology involves The use of living organisms (often, but not always, microorganisms) to produce a product. Sometimes involves recombinant DNA. The product may be isolated from the organism the genetically changed organism itself

11. The larger coho salmon contain a genetically engineered gene for growth hormone.

12. The yellow rice have been genetically engineered to produce beta carotene. Source of Vitamin A.

13. Definitions Computational biology Biology problem-solving done with the use of a computer Biology in silico When applied to molecular biology, called bioinformatics

14. What is the National Center for Biotechnology Information Established in 1988 as a national resource for molecular biology information, NCBI is financed by the US government.NCBI Its purposes are to create public databases accessible by computer conduct research in computational biology develop software tools for analyzing genome data, disseminate biomedical information online - all for the better understanding of molecular processes affecting human health and disease.

15. Molecular biology moves in many directions in the lab. in silico  in vitro  in vivo

16. Cooperative Analysis Computer Analysis omputer Results Biological Insight Biological Data Experiments Computer Analysis Biological Data Computer Results

17. 2 Areas of Early Basic Research that Pushed Molecular Biology Ahead Knowledge of small genomes that are propagated in bacteria Plasmids Phage Knowledge of microbial restriction systems Restriction endonucleases are used in cloning with both plasmids and phage

18. What is a restriction system, and how has knowledge of restriction systems advanced gene manipulation?

19. Restriction Systems provide microorganisms with resistance to invading organisms or foreign DNA. consist of a related pair of enzymes Endonuclease that cuts foreign DNA Methylase that protects host DNA

21. Restriction Endonuclease (RE) Endonuclease = enzyme that cuts within nucleic acid strands REs cut within double-stranded (ds) DNA

22. Restriction Endonuclease (RE) There are hundreds of different REs from different microorganisms Each RE cuts DNA at a specific “recognition sequence” of nucleotides. Examples: EcoRI– G’AATTC; AluI – AG’CT Each recognizes its specific “recognition sequence” and cuts both strands of DNA wherever that sequence is found, but nowhere else.

25. Methylase Adds a methyl group to a base within the same recognition sequence as its corresponding RE. Me  GAATTC CTTAAG  Me

26. Even though the sequence is the recognition sequence for the corresponding RE, that RE can not cut the sequence if it is appropriately methylated.

27. Methylase adds methyl groups during replication of the bacterial genome. Foreign DNA comes in unmethylated and is cut before it can be methylated. What is missing from the pictures of Part a.

28. What is the technological utility of an RE in vitro? Allows investigator to digest (cut) DNA in vitro at a specific site(s) and leave known ends. Results of digestion with a given enzyme will be reproducible from one repeated experiment to the next. The results of the RE digest can be predicted. if the sequence of the DNA to be cut is known and if the recognition sequence of the enzyme is known, or if the digest has been done on the same DNA before and a restriction map has been drawn.

29. Restriction Map of Vectors: pBR322 & pGEM ® -3Z These maps show only those enzymes that cut the plasmid in only one place.

30. REs Types I, II, III Type II is the only type widely used type for gene manipulation. Recognition sequence is usually “palindromic”. Palindrome = a word that reads the same backwards and forwards. Examples: TAT, OTTO, but for DNA, the sequence in both strands must be considered to recognize the presence of a palindrome. Each enzyme always cuts at the same single location within or at the very edge of the recognition sequence, depending upon the enzyme.

31. REs can produce Blunt ends Sticky ends 3’ recessed = 5’ overhang 5’ recessed = 3’ overhang

32. Two different REs may be Isoschizomers Cut at the same recognition sequence Isocaudamers Cut at different recognition sequences, but leave the same tail (overhang) What is the tailfin of a fish called? (Fragments of DNA with the same tails can be pieced together [ligated].)