DNA Technology & Genomics Chapter 20. Biotechnology: DNA Technology & Genomics

The BIG Questions… How can we use our knowledge of DNA to: diagnose disease or defect? cure disease or defect? change/improve organisms? What are the techniques & applications of biotechnology? direct manipulation of genes for practical purposes

Biotechnology Genetic manipulation of organisms is not new humans have been doing this for thousands of years plant & animal breeding INDIRECTLY manipulating DNA

Evolution & breeding of food plants Evolution of Zea mays from ancestral teosinte (left) to modern corn (right). The middle figure shows possible hybrids of teosinte & early corn varieties

Animal husbandry / breeding

Biotechnology today Genetic Engineering Our tool kit… manipulation of DNA if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with this unit is a survey of those tools… Our tool kit…

Bioengineering Tool kit Basic Tools restriction enzymes ligase plasmids / cloning DNA libraries / probes Advanced Tools PCR DNA sequencing gel electrophoresis Southern blotting microarrays

Cut, Paste, Copy, Find… Word processing metaphor… cut paste copy find restriction enzymes paste ligase copy plasmids bacteria transformation PCR find Southern blotting / probes

Cut DNA Restriction enzymes restriction endonucleases discovered in 1960s evolved in bacteria to cut up foreign DNA (“restriction”) protection against viruses & other bacteria bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA

Restriction enzymes Madam I’m Adam Action of enzyme cut DNA at specific sequences restriction site symmetrical “palindrome” produces protruding ends sticky ends Many different enzymes named after organism they are found in EcoRI, HindIII, BamHI, SmaI CTGAATTCCG GACTTAAGGC  CTG|AATTCCG GACTTAA|GGC 

Biotech use of restriction enzymes GAATTC CTTAAG DNA Restriction enzyme cuts the DNA Sticky ends (complementary single-stranded DNA tails) AATTC G AATTC G G CTTAA G CTTAA Add DNA from another source cut with same restriction enzyme AATTC G G AATTC CTTAA G DNA ligase joins the strands. Recombinant DNA molecule GAATTC CTTAAG

Paste DNA Sticky ends allow: Ligase H bonds between complementary bases to anneal Ligase enzyme “seals” strands bonds sugar-phosphate bonds covalent bond of DNA backbone

Copy DNA Plasmids small, self-replicating circular DNA molecules insert DNA sequence into plasmid vector = “vehicle” into organism transformation insert recombinant plasmid into bacteria bacteria make lots of copies of plasmid grow recombinant bacteria on agar plate clone of cells = lots of bacteria production of many copies of inserted gene DNA  RNA  protein  trait

Recombinant DNA movie Gene cloning

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

    Cut, Paste, Copy, Find… Word processing metaphor… cut paste restriction enzymes paste ligase copy plasmids bacteria transformation PCR find Southern blotting / probes    

Electrophoresis & RFLPs Southern Blot, PCR, Advanced Techniques Electrophoresis & RFLPs Southern Blot, PCR,

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”

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 PCR 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."

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.

Any Questions??

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?

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