Advanced Techniques Electrophoresis, PCR, Southern Blot, Sequencing, RFLPs, Microarrays 1
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 2
Gel Electrophoresis 3
Gel Electrophoresis 4
Measuring fragment size compare bands to a known standard usually lambda phage cut with HindIII nice range of sizes with a distinct pattern 5
RFLP Restriction Fragment Length Polymorphism change in DNA sequence affects restriction enzyme cut site will create different band pattern 6
Southern Blot Want to locate a sequence on a gel? 7
Southern blot Transfer DNA from gel to filter paper hybridize filter paper with tagged probe fragment with matching sequence lights up 8
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! 9
PCR primers The primers are critical! need to know sequence to make proper primers primers flank target sequence start with long piece of DNA & copy a specified shorter segment primers define section of DNA to be cloned Taq polymerase from hot springs bacteria why do we use it? play DNAi movie 20-30 cycles 3 steps/cycle 30 sec/step 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 contraband Taq clones, so scientists could grow up their own bacteria to produce Taq in the lab. It s like pirated software -- pirated genes! 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. 10
1985 1993 Kary Mullis 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." 11
DNA Sequencing Sanger method determine the nucleotide sequence of DNA synthesize comple mentary strand of DNA in vitro use tagged bases ddntp dideoxynucleotides 12
Dideoxynucleotides reagent mix for DNA replication normal N-bases dideoxy N-bases missing O for bonding of next nucleotide terminates chain radioactively tagged DNA polymerase primer buffers & salt play Sequencing movie 13
Reading the sequence Load gel with sequences from dda, ddt, ddc, ddg in separate lanes read lanes manually & carefully polyacrylamide gel 14
Fred Sanger This was his 2nd Nobel Prize!! 1st was in 1958 for the protein structure of insulin 1978 1980 15
Advancements to sequencing Fluorescent tagging no more radioactivity all 4 bases in 1 lane each base a different color Automated reading 16
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 17
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 18
Human Genome Project U.S government project 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 19
Different approaches map-based shot gun 20
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 21
GenBank Database of genetic sequences gathered from research Publicly available! 22
The Progress 4.E+10 3.E+10 3.E+10 2.E+10 first metazoan complete (flatworm) 122+ bacterial genomes 2.E+10 1.E+10 5.E+09 0.E+00 Dec-82 # of DNA base pairs (billions) in GenBank Dec-83 Data AP from Biology NCBI and TIGR (www.ncbi.nlm.nih.gov and www.tigr.org ) Dec-84 Dec-85 first eukaryote complete (yeast) First 2 bacterial genomes complete Dec-86 Dec-87 Dec-88 Dec-89 Dec-90 Dec-91 Dec-92 Dec-93 Dec-94 Dec-95 Dec-96 Dec-97 Dec-98 Dec-99 Dec-00 Official 15 year Human Genome Project: 1990-2003. Dec-01 Dec-02 Jun-03 17 eukaryotic genomes complete or near completion including Homo sapiens, mouse and fruit fly S1 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!! 23
Other genomes 24
Microarrays Measuring expression of genes in a tissue sample samples of mrna from cells make cdna 2-color fluorescent tagging hybridize to spots of genes colored spots = gene expression red, green, yellow 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. 25
When to use microarrays? Research tool it s all about comparisons gene expression. before vs. after treatment cancer vs. normal cells wound healing vs. scarring stages of development color coding red = expression in 1 sample green = expression in other sample yellow = expression in both samples dark = low expression in both 26
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. 27
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! 28
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. 29
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? 30