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PCR Review

PCR Review. Collect Lab Write-ups What is PCR ? What are the 3 steps essential for amplification? What are the essential reagents/ingredients? PCR Amplification: www.hhmi.org/biointeractive. 3 Essential Steps.

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PCR Review

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  1. PCR Review • Collect Lab Write-ups • What is PCR? • What are the 3 steps essential for amplification? • What are the essential reagents/ingredients? • PCR Amplification: www.hhmi.org/biointeractive

  2. 3 Essential Steps • Step 1: Melt – Separate the two DNA chains in the double helix by heating the vial containing the PCR reaction mixture to 95°C for 30 seconds. • Step 2: Anneal – The primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 60°C. At this temperature, the primers bind (anneal) to the single-stranded DNA. • Step 3: Extend – is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70°C for 45 seconds.

  3. Essential Reagents • Original Sample DNA • Large quantities of oligonucleotide DNA primers (Anneal) • Primers are small pieces of DNA that bind to specific sequences • Heat-stable DNA polymerase that extends the copy DNA strand. (Extend) • Large quantities of the four nucleotides adenine, cytosine, guanine, and thymine (Extend)

  4. PCR • Amplifies a DNA when only small sample of DNA • Resulting multiple copies of DNA can be manipulated in various lab applications – DNA Fingerprinting, DNA Sequencing

  5. DNA Sequencing • Virtually all the information required for growth and development of an organism is encoded in the DNA of its genome. • DNA restriction enzymes can break up nucleotides into restriction fragments a few hundred bases long. • PCR can help amplify DNA and make multiple copies • This has led to the need for development of methods to determine the exact sequence of stretches of DNA • Currently we are only able to accurately sequence around 600 bases accurately – great start, but only a start. • Human genome is over 3 Billion bases long, arranged on 23 chromosomes.

  6. How do we find out what is the exact sequence of bases in a DNA fragment? • Sanger Method utilized in Cycle Sequencing • Many copies of DNA are made in a Thermocycler • Added to a reaction mixture: • that contains normal deoxynucleotides (A, C, G, and T) • special dideoxynucleotides (A*, C*, G*, and T*) that are tagged with fluorescent markers. The fluorescent markers differ for each base, and are designed to fluoresce with different colors (G* is yellow, T* is green, for example) • Utilizing the Sanger method you terminate the replication process at random places with dideoxynucleotides so the copies are all partial sequences with different lengths. • Dideoxynucleotides - http://www.dnalc.org/ddnalc/resources/sangerseq.html

  7. Dideoxynucleotide (didNTP)

  8. Dideoxynucleotide – didNTP • Terminates replication • Dideoxynucleotides can substitute for normal deoxynucleotides during replication • when such a substitution occurs by chance, that chain can no longer be extended, terminating that DNA strand. • Dideoxynucleotides are thus called terminators.

  9. Gel Electrophoresis

  10. Automated sequencing gel

  11. Computer Sequencing Output

  12. Adding all 4 deoxynucleotides (dNTP) into the picture (DID NOT GO OVER IN CLASS – OMIT)

  13. Human Genome Project • Effort to Map the entire human genome • National Institute of Health and National Science Foundation have funded the creation of libraries of gene maps. • Researchers use restriction enzymes to break the DNA into a number of identifiable fragments • Small number of Human Genes • 30-40,000 genes. Only 2 or 3 times the number found in the fruit fly and nematode worm. • Many genes of disparate organisms are turning out to be very similar. • Through comparative analysis genes of other species help scientists interpret human data.

  14. Challenges of Human Genome • Finding exact DNA sequences is time intensive • The Genome is enormous – Approximately 3 Billion nucleotide pairs of DNA • Only 35,000 genes have been identified as protein coding or genes for tRNA and rRNA. • About 97% of the total human DNA is noncoding DNA – “Junk DNA” • Junk DNA does not code for proteins – we do not yet understand its functions.

  15. Junk DNA • Includes Introns and non-coding DNA located between genes • Much of the DNA between genes consists of repetitive DNA • Such repetitions found at the centromeres and ends of chromosomes, suggest this DNA plays a role in chromosome structure • Telomeres – are repetitive DNA found at the ends of chromosomes may have a protective function.

  16. Human Genome Homework • Read Human Genome Project (HGP) Articles • In notebook • List 5 benefits of the HGP • List 3 challenges of the HGP • Identify 2 ethical issues with genomics research.

  17. Potential Benefits of Human Genome Project • Insight into the mysteries of embryonic development and evolution • Will aid in the diagnosis, treatment, and possible prevention of common ailments like heart disease, allergies, diabetes, etc. • Ability to identify hundreds of disease-associated genes—Alzheimer’s and cancer. • By transferring a gene of interest into a bacterial vector useful proteins can be made. • Protein products of Recombinant DNA Technology: • Human Insulin • Hepatitis B vaccine • Erythropoietin (EPO)

  18. LosatSoy • What was this article about? • What health risk were they trying to address? • What are some other potentially beneficial GM foods.

  19. ACME FOODS INC. • Start Lab.

  20. ACME FOODS INC. • Which product would you produce? Why did you choose that product? • What are the concerns or issues that this product might bring up? Who are the people involved in this issue? • Who will do the testing for your product? What type of testing needs to be done? • Will you label your food product? Why or Why not? • How will you promote your product to consumers? • As a consumer, how would you make a decision about whether to purchase this product? • Would you want GM food labels ?

  21. Genetically Modified (GM) Foods • Human Population is growing exponentially • Currently about 6.5 Billion people on the planet. • In the next 50 years we will be over 9 Billion. • Concern will be how will we feed the growing human population? • GM Organisms – acquire one or more genes by artificial means rather than by traditional breeding methods.

  22. GM Plants • Ti Plasmid is utilized as a vector • Gene of interest is inserted to form a recombinant plasmid and introduced into plant cells • If the newly acquired gene is from another species, the recombinant organism is called a transgenic organism. • US Department of Agriculture estimates of genetically modified crops: • 74 % of American soybean crop • 32% of the corn crop • 71% of the cotton crop

  23. GM Benefits • Herbicide resistance • Microbe resistance • Pest resistance • Improved Nutritional Content • Golden Rice • LosatSoy • Your Product?

  24. Could GM Organisms harm human health or the environment? • Transferring of New Types of Allergens in GM foods • Transgenic plants may pass their genes to close relatives in nearby wild areas. • Could produce “Superweeds.”

  25. Case Study – The monarch butterfly • Monarchs migration paths can span up to 3,000 miles long. • They travel through forests and over fields, including more and more acres growing Bt corn. • Bt Corn has been genetically modified to produce a pesticide. • Pesticide is found in all parts of the plant – including pollen • Pollen can drift off onto leaves of nearby milkweed plants. • Milkweed plants are the favorite food for Monarch caterpillars. • Some researchers claimed the Bt corn stunted or killed the monarch caterpillars

  26. Leroy Hood • “What science does is give society opportunities. What we have to do is look at these opportunities and then set up the constraints and the rules that will allow society to benefit in appropriate ways.”

  27. Issues • What did Leroy Hood mean? • What are the problems with being able to modify DNA? • Who should have control over genetic information?

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