1. Bioinformatics & Biotechnology Lecture 1 Sequencing BLAST PCR Gel Electrophoresis

2. Bioinformatics/Computational Biology New & exciting field in Biology If you love biology and computers (AKA you’re a geek), you’ll love it! Especially HUGE in molecule biology Way of storing and organizing massive amount of biological data Computers are a MAJOR tool in the biological sciences

3. We just learned about DNA Replication… Scientists have discovered 2 million living species on this planet Almost 2 million types of viruses that each have a genome Genomes can be millions or billions of base pairs in length We can now sequence DNA…where can we put all the information?! How can we access and use the information other scientists have discovered?

4. What we can use large databases of DNA sequences for… Forensics Identification Paternity tests Comparative biology Taxonomy But first…how do we sequence a genome? Let’s learn about PCR first!

5. Polymerase Chain Reaction (PCR) Probably the single most important piece of molecular technology we have today In a nutshell, it allows us to make thousands of copies of one portion of DNA from just a single sample! Done in a thermocycler

6. The Five PCR Ingredients Buffer (aqueous with ions) Template DNA Taq polymerase (thermas aqueous) Forward and reverse primers Nucleotides (dNTPs)

7. Three Steps of PCR Denaturation Annealing Extension

8. Now we have all this DNA…what to do? ‘Digest’ the DNA with restriction enzymes Gel Electrophoresis – separates DNA fragments by size Gel matrix made of agarose – seaweed extract

9. Gel Electrophoresis Must do PCR first. Each “band” is thousands of fragments of the same size Smaller fragments get to bottom first DNA travels through agarose gel because of electric current running though it (remember, DNA is negatively charged) Can determine fragment length based on ladder (standard of known lengths) Used at crime scenes, paternity tests, gene amplification validation http://www.pbs.org/wgbh/nova/education/body/create-dna- fingerprint.html

10. Uses of PCR + Sequencing Rapid exponential amplification of specific DNA sequences Allows scientists to study specific pieces of DNA (primer design) Taxonomy Transcriptomics (what genes get expressed at what times) Crime scene investigation Paternity tests Bacterial/viral infection identification Heredity

11. DNA Sequencing – Sanger Method Ingredients Buffer DNA template (what you’re trying to sequence) A universal primer 90% dNTP’s (deoxyribonucleotide triphosphates) 10% ddNTP’s (dideoxy….) with fluorescent tags DNA polymerase

12. Sanger Sequencing ddNTPs terminate elongation of DNA strand Each ddNTP is labeled differently (with a different color fluorescence) End up with thousands of fragments of different lengths Put the reaction mixture in a gel http://www.dnalc.org/resources/animations/cycseq.html

13. Sanger Sequencing Sanger sequencing machine has a sensor at the bottom that reads the fluorescence of each separated band Each color indicates a letter Can put together in a graph and read it  sequence known! http://www.dnalc.org/resour ces/animations/cycseq.html

14. Once Sequenced… Add it to NCBI’s BLAST library Scientists all over the world collect, share, and collaborate on sequence data (along with other types of data) put on this website

15. A cool use of PCR/Sequencing Sam Wasser’s Team Comparing sequencing of poached elephant populations to study how elephant behavior/family dynamics has been effected by family-unit disruption Sequencing as many elephants in Africa as possible (via poop) and ivory found on the black market to pinpoint poaching hot spots (can be used with any endangered species)