Electrophoresis & RFLPs Advanced Techniques Electrophoresis & RFLPs Modified from: Kim Foglia, Explore Biology

Gel Electrophoresis Separation of DNA fragments by size DNA is negatively charged moves toward + charge in electrical field agarose gel “swimming through Jello” smaller fragments move faster cut DNA with restriction enzymes

Gel Electrophoresis

Gel Electrophoresis

Measuring fragment size compare bands to a known “standard” usually lambda phage virus cut with HindIII nice range of sizes with a distinct pattern

RFLP Restriction Fragment Length Polymorphism differences in DNA between individuals change in DNA sequence affects restriction enzyme “cut” site will create different band pattern

Polymorphisms in populations Differences between individuals at the DNA level

RFLP use in forensics 1st case successfully using DNA evidence 1987 rape case convicting Tommie Lee Andrews “standard” semen sample from rapist blood sample from suspect “standard” “standard” semen sample from rapist blood sample from suspect “standard”

RFLP use in forensics Evidence from murder trial Do you think suspect is guilty? blood sample 1 from crime scene blood sample 2 from crime scene blood sample 3 from crime scene “standard” blood sample from suspect blood sample from victim 1 blood sample from victim 2 “standard”

Modified from: Kim Foglia, Explore Biology Any Questions?? Modified from: Kim Foglia, Explore Biology

RFLP use in forensics Evidence from murder trial Do you think suspect is guilty? blood sample 1 from crime scene blood sample 2 from crime scene blood sample 3 from crime scene “standard” blood sample from suspect blood sample from victim 1 blood sample from victim 2 “standard”

Advanced Techniques Part 2 Southern Blot, PCR, Sequencing, Human Genome Project Modified from: Kim Foglia, Explore Biology

Southern Blot Want to locate a sequence on a gel?

Southern blot Transfer DNA from gel to filter paper hybridize filter paper with tagged probe fragment with matching sequence “lights up”

Hybridization in Southern Blotting Use radioactive probe to locate gene on filter paper go back to gel & cut out piece of DNA you want to collect

Polymerase Chain Reaction (PCR) What if you have too little DNA to work with? PCR is a method for making many copies of a specific segment of DNA ~only need 1 cell of DNA to start copying DNA without bacteria or plasmids!

PCR process It’s copying DNA in a test tube! What do you need? template strand DNA polymerase enzyme nucleotides primer Thermocycler

What does 90°C do to our DNA polymerase? PCR process What do you need to do? in tube: DNA, enzyme, primer, nucleotides heat (90°C) DNA to separate strands (denature) cool to hybridize (anneal) & build DNA (extension) What does 90°C do to our DNA polymerase?

PCR primers The primers are critical! need to know a bit of sequence to make proper primers primers bracket target sequence start with long piece of DNA & copy a specified shorter segment primers define section of DNA to be cloned PCR is an incredibly versatile technique: An important use of PCR now is to “pull out” a piece of DNA sequence, like a gene, from a larger collection of DNA, like the whole cellular genome. You don’t have to go through the process of restriction digest anymore to cut the gene out of the cellular DNA. You can just define the gene with “flanking” primers and get a lot of copies in 40 minutes through PCR. Note: You can also add in a restriction site to the copies of the gene (if one doesn’t exist) by adding them at the end of the original primers. 20-30 cycles 3 steps/cycle 30 sec/step

The polymerase problem PCR 20-30 cycles 3 steps/cycle 30 sec/step Heat DNA to denature it 90°C destroys DNA polymerase have to add new enzyme every cycle almost impractical! Need enzyme that can withstand 90°C… Taq polymerase from hot springs bacteria Thermus aquaticus Taq = Thermus aquaticus (an Archaebactera) Highly thermostable – withstands temperatures up to 95°C for more than 40min. BTW, Taq is patented by Roche and is very expensive. Its usually the largest consumable expense in a genomics lab. I’ve heard stories of blackmarket Taq clones, so scientists could grow up their own bacteria to produce Taq in the lab. It’s like pirated software -- pirated genes! play DNAi movie

Kary Mullis 1985 | 1993 development of PCR technique a copying machine for DNA In 1985, Kary Mullis invented a process he called PCR, which solved a core problem in genetics: How to make copies of a strand of DNA you are interested in. The existing methods were slow, expensive & imprecise. PCR turns the job over to the very biomolecules that nature uses for copying DNA: two "primers" that flag the beginning & end of the DNA stretch to be copied; DNA polymerase that walks along the segment of DNA, reading its code & assembling a copy; and a pile of DNA building blocks that the polymerase needs to make that copy. As he wrote later in Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents and a source of heat. The DNA sample that one wishes to copy can be pure, or it can be a minute part of an extremely complex mixture of biological materials. The DNA may come from a hospital tissue specimen, from a single human hair, from a drop of dried blood at the scene of a crime, from the tissues of a mummified brain or from a 40,000-year-old wooly mammoth frozen in a glacier."

DNA Sequencing Sanger method determine the base sequence of DNA dideoxynucleotides ddATP, ddGTP, ddTTP, ddCTP missing O for bonding of next nucleotide terminates chain

DNA Sequencing Sanger method 1 Sanger method synthesize complementary DNA strand in vitro in each tube: “normal” N-bases dideoxy N-bases ddA, ddC, ddG, ddT DNA polymerase primer buffers & salt 2 3 4 2

Reading the sequence Load gel with sequences from ddA, ddT, ddC, ddG in separate lanes read lanes manually & carefully polyacrylamide gel

Fred Sanger 1978 | 1980 This was his 2nd Nobel Prize!! 1st was in 1958 for the structure of insulin

Advancements to sequencing Fluorescent tagging no more radioactivity all 4 bases in 1 lane each base a different color Automated reading

Advancements to sequencing Fluorescent tagging sequence data Computer read & analyzed

Advancements to sequencing Capillary tube electrophoresis no more pouring gels higher capacity & faster Applied Biosystems, Inc (ABI) built an industry on these machines 384 lanes

Big labs! economy of scale PUBLIC Joint Genome Institute (DOE) MIT Washington University of St. Louis Baylor College of Medicine Sanger Center (UK) PRIVATE Celera Genomics Celera: Rockville, MD & San Francisco, CA Baylor: Houston TX

Automated Sequencing machines Really BIG labs!

Human Genome Project U.S government project Celera Genomics begun in 1990 estimated to be a 15 year project DOE & NIH initiated by Jim Watson led by Francis Collins goal was to sequence entire human genome 3 billion base pairs Celera Genomics Craig Venter challenged gov’t would do it faster, cheaper private company 1990-1995 build the technology groundwork improve sequencing methods build clones build better data management systems (computer tools to find overlaps) better, cheaper, faster! 1996-1998 painstaking sequencing work 1998 Celera genomics challenge 2000 rough draft of human genome (90% sequence, 99% accurate) 2001 1st draft of human genome 2003 “finished” sequence of human genome can’t sequence telomeres & centromeres

Different approaches “map-based method” “shotgun method” gov’t method Craig Venter’s method Cut DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments. 2. Cut and clone into smaller fragments. 1. Cut DNA entire chromosome into small fragments and clone. 2. Sequence each segment & arrange based on overlapping nucleotide sequences. 3. Assemble DNA sequence using overlapping sequences.

Human Genome Project On June 26, 2001, HGP published the “working draft” of the DNA sequence of the human genome. Historic Event! blueprint of a human the potential to change science & medicine

Sequence of 46 Human Chromosomes 3G of data 3 billion base pairs

Raw genome data

GenBank Database of genetic sequences gathered from research Publicly available!

Organizing the data

Maps of human genes… Where the genes are… mapping genes & their mutant alleles

And we didn’t stop there… The main imperative for a primate genome project is to find out why humans develop certain diseases that other primates are immune to — like HIV.

The Progress # of DNA base pairs (billions) in GenBank 122+ bacterial genomes first metazoan complete (flatworm) first eukaryote complete (yeast) 17 eukaryotic genomes complete or near completion including Homo sapiens, mouse and fruit fly First 2 bacterial genomes complete In the last 5 years or so the amount of data has grown exponentially, including the growth of online databases and resources. In this slide we have the growth of the total number of base pairs and the total number of genomes completed since the beginning of the Human Genome Project. As you can see, the sheer number and growth of this resource has been impressive—and daunting—in the last 5 years. Few scientists are aware of, or make full use of, all the open-source and public resources available to them through the internet. The Annual Nucleic Acids Research Database issue listing contained 548 databases this year!! And, as this quote mentions, only half of those who use the databases are familiar with their tools. This Wellcome Trust study also made it clear that many people become users of a database after being told about it by colleagues. “Despite the large amount of publicity surrounding the Human Genome Project, a recent survey conducted on behalf of the Wellcome Trust indicates that only half of biomedical researchers using genome databases are familiar with the tools that can be used to actually access the data. In “The Molecular Biology Database Collection: 2003 update” by Andreas D. Baxevanis in the Jan 1, 2003 NAR database issue. # of DNA base pairs (billions) in GenBank Official “15 year” Human Genome Project: 1990-2003. Data from NCBI and TIGR (www.ncbi.nlm.nih.gov and www.tigr.org )

How does the human genome stack up? Organism Genome Size (bases) Estimated Genes Human (Homo sapiens) 3 billion 30,000 Laboratory mouse (M. musculus) 2.6 billion Mustard weed (A. thaliana) 100 million 25,000 Roundworm (C. elegans) 97 million 19,000 Fruit fly (D. melanogaster) 137 million 13,000 Yeast (S. cerevisiae) 12.1 million 6,000 Bacterium (E. coli) 4.6 million 3,200 Human Immunodeficiency Virus (HIV) 9700 9

What have we found? When you go looking…

…you will certainly find something!

Modified from: Kim Foglia, Explore Biology Any Questions?? Modified from: Kim Foglia, Explore Biology

Modified from: Kim Foglia, Explore Biology Advanced Techniques Microarrays Modified from: Kim Foglia, Explore Biology

Where do we go next…. DNA RNA protein trait When a gene is turned on, it creates a trait want to know what gene is being expressed extract mRNA from cells mRNA = active genes How do you match mRNA back to DNA in cells???

slide with spots of DNA each spot = 1 gene Microarrays Create a slide with a sample of each gene from the organism each spot is one gene Convert mRNA  labeled cDNA mRNA  cDNA mRNA from cells reverse transcriptase

Microarrays Labeled cDNA hybridizes with DNA on slide slide with spots of DNA each spot = 1 gene Microarrays Labeled cDNA hybridizes with DNA on slide each yellow spot = gene matched to mRNA each yellow spot = expressed gene cDNA matched to genomic DNA mRNA  cDNA Developed by Pat Brown at Stanford in late 1980s Realized quickly he needed an automated system: robot spotter Designed spotter & put plans on Internet for benefit of scientific community.

Application of Microarrays 2-color fluorescent tagging Comparing treatments or conditions = Measuring change in gene expression sick vs. healthy; cancer vs. normal cells before vs. after treatment with drug different stages in development Color coding: label each condition with different color red = gene expression in one sample green = gene expression in other sample yellow = gene expression in both samples black = no or low expression in both It’s all about comparisons! Powerful research tool.

“DNA Chip” Patented microarray technology from Affymetrix automated DNA synthesis of genes of interest on chip chips are more consistent smaller spots/more spots per chip can buy specific chips human chip mouse chip etc. chip = $1000 machine to read chip = $150,000

Biotechnology today: Applications Application of DNA technologies basic biological research medical diagnostics medical treatment (gene therapy) pharmaceutical production forensics environmental cleanup agricultural applications …and then there’s the ethics issues!

Application of recombinant DNA Combining sequences of DNA from 2 different sources into 1 DNA molecule often from different species human insulin gene in E. coli (humulin) frost resistant gene from Arctic fish in strawberries “Roundup-ready” bacterial gene in soybeans BT bacterial gene in corn jellyfish glow gene in Zebra “Glofish” In 1978, scientists at the Biotechnology Company, Genentech, cloned the gene for Human Insulin. Genentech licensed the human insulin technology to Eli Lilly, where it was named "Humulin" or Recombinant Human Insulin. In 1982, human insulin became the first recombinant DNA drug approved by FDA. Today, Humulin is made in Indianapolis in gigantic fermentation vats, 4 stories high and filled with bacteria!!! These fermentation vats operate 24 hours a day, year round. The human insulin protein made by the E. coli bacteria is collected from the vats, purified, and packaged for use by patients with diabetes.

Human Cloning Human cloning is very controversial & not the main goal of biotechnology

Modified from: Kim Foglia, Explore Biology Any Questions?? Modified from: Kim Foglia, Explore Biology

What next? After you have cloned & amplified DNA (genes), you can then tackle more interesting questions how does gene differ from person to person? …or species to species is a certain allele associated with a hereditary disorder in which cells is gene expressed? where is gene in genome?