Bioinformatics & Biotechnology

Lecture 1Sequencing

BLASTPCR

Gel Electrophoresis

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

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?

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!

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

The Five PCR Ingredients

• Buffer (aqueous with ions)• Template DNA• Taq polymerase (thermas aqueous)• Forward and reverse primers • Nucleotides (dNTPs)

Three Steps of PCR

• Denaturation• Annealing• Extension

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

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

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

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

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

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/

resources/animations/cycseq.html

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

A cool use of PCR/SequencingSam 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)