This "central dogma" of today's molecular biology has applications for identifying genetic biodiversity and disorders, as well as corpses and suspects and babies switched at birth. 
(See http://www.wired.com/wired/archive/10.11/dna.html) 

Genetic engineering and gene therapy are other applications of the "central dogma" in which we create different proteins by changing the DNA. Phenotypes are produced by

1. proteins
2. products of chemical reactions catalyzed by proteins
3. regulation of the timing and location (which cells in which organs?) of replication, transcription, and translation.

Can we change phenotypes by changing the genotype, the DNA? Should we? Who should decide?

Tools, part 1:  Preview of some tools (Wednesday lecture)

Genome Tools (Friday, 11 Oct) 
         (Friday's lecture)

Medical Tools (Monday 14 Oct)
 

Other Applications (Wednesday, 16 Oct)    
       
Links to Important Web sites

BIO 103 HOME PAGE

"TOOLS, part 1":  For each, know what it does or why anybody would use it and at least a baby explanation of how or why it works especially if it involves something relevant to the natural structure and function of DNA. In your textbook, pay particular attention to the "technical detail" parts of figures.  Most of these tools are in chapters not assigned this semester or in chapters we have already finished.

1. Restriction  enzymes  are nucleases; they cut DNA or RNA into fragments ( Box  15.1, p.302).  (In bacterial cells, the natural function of restriction enzymes is to destroy the nucleic acids of enemies.)  Also see picture from last year's textbook.  EcoRI is the most famous restriction enzyme, but there are at least a hundred different restriction enzymes, each of which attacks a different palindromic sequence.  Restriction enzymes are used to create cuts and fragments for DNA analysis and also for genetic engineering.
   • http://vector.cshl.org/dnaftb/24/animation/fs.html  
   • restriction enzyme structure http://www.clunet.edu/BioDev/omm/bamh1/molmast.htm 
2. Fragments produced by restriction enzymes have different lengths.  Fragments are sometimes called RFLPs (pronounced "riff-lips"), especially when the different lengths reflect differing alleles or at least individual differences. 
3. Electrophoresis separates and sorts fragments according to their lengths, box 15.2, p. 308.   Electrophoresis and autoradiography (Fig.12.12c) are explained in more detail on p. 50 (Box 3.2).Want to see RFLPs eletrophoresed?  
   • Animation (with Southern blot and forensic evidence analysis at end:  http://vector.cshl.org/Shockwave/southan.html; requires  plug-in (download linked)
   • also http://vector.cshl.org/dnaftb/24/animation/fs.html   
   • a new alternative separation method for DNA http://www.news.cornell.edu/releases/Sept02/entropicSeparation.ws.html 
4. Probes are oligonucleotides, short pieces of RNA or single-stranded DNA.  Probes work by hybridizing with single strands of DNA or RNA; it's called hybrid (new meaning) because the probe and its target are from different critters.  The probe and its target have complementary base sequences; so they stick together by hydrogen bonds between the complementary pairs, just like a primer will stick to DNA in replication or transcription.  Radioactive or fluorescent probes are especially useful in research.  Can you figure out why?  
   • A probe can ID specific base sequences on the electrophoresis gel or on blots (box 15.2, p. 309) which absorb the fragments from the gel. 
   • A probe can also pinpoint the location of a gene on a chromosome (as you will see in CD activity 16.1) or to find which colonies of genetically engineered bacteria have the right DNA sequence (as you will see in CD activity 17.1).
   • Probes can also be used in vivo; one example is in situ hybridization which is used to tell which genes are active in specific tissues.  The mRNAs which have been transcribed can be detected with radioactive or fluorescent DNA probes (Box 19.1). 
   • probes have other uses we'll be seeing next week, like in    Finding the Growth Hormone Gene (probe, complementary base pairing, making a probe, radioactive labels)  
5. PCR, fig. 12.11, uses primers and polymerase and temperature cycles to replicate DNA in huge quantities.  
   • PCR DNA copy-making:  study pp. 240b-242a  (especially fig. 12.11a, 12.11b, 12.11b continued ) and CD  Activity 12.2 Polymerase Chain Reaction (including vocabulary:  primer, polymerase,…etc.).  
   • See animation (http://vector.cshl.org/Shockwave/pcranwhole.html; requires Shockwave plug-in).
   • WHAT IS PCR?  
6. How can we sequence a gene?  =What is the base sequence of this DNA? (fig. 12.12)   DNA base sequence determination:  The Sanger (dideoxy) procedure is still used in some professional laboratories today, even though there are machines which determine the base sequence faster.  The newer procedures are based on the same principles as dideoxy sequencing described on p. 242.    Study fig 12.12. and these CD activities:  Sequencing the DNA and Finding Genes in the Sequence (shotgun, …)
   Here are some other sources:

• sequencing animation:  http://vector.cshl.org/Shockwave/cycseq.html)

• http://vector.cshl.org/dnaftb/23/animation/fs.html   #23 shows the didNTP method and also demonstrates redundancy of the genetic code
• newer sequencing methods http://www.genome.gov/Pages/EducationKit/online.htm   
•  get Celera to do it for you.  Can I buy my own sequencer?  Or software to analyze my gels or blots?  

• and this just in:  differing rates of light-controlled oxidation in bases can lead to new methods of sequencing and possible therapy http://www.eurekalert.org/pub_releases/2002-10/dnl-ttl100402.php 

7. Ligase enzyme can make recombinant DNA  for genetic engineering or GM (genetic modification).  Ligase "glues"  fragments together, usually into circles of DNA, called plasmids (which are natural) or BACs (bacterial artificial chromosomes).   (Recombinant DNA is not expressed (cannot work) unless it is adjacent to promoter DNA segments, fig. 13.5, 13.6.) (In live normal cells, the natural function of ligase is to combine the Okazaki fragments of the lagging strand in DNA replication, fig. 12.9 #7) If you want to know more, work ahead on  Creating a cDNA Library (good review of reverse transcriptase, cDNA, restriction endonuclease, complementary base pairing, ligase, plasmid, library)  
8. DNA can be synthesized in the lab, but synthetic DNA is used mostly as probes or sometimes as deliberate mutations, tiny inserts into recombinant DNA.   (DNA used for genetic engineering is always recombinant DNA, involving genes from natural sources, partly because real genes are too long to be synthesized practically and partly because the proteins which would be translated do not fold into functional and predictable tertiary shapes. Scientists don't know enough about proteins yet.)  You don't have to know how they synthesize DNA.   You can buy synthetic DNA at www.operon.com, among other places.

GENOME TOOLS (Assignment for Friday)

• What did the HUMAN GENOME PROJECT (HGP) do?  
  • Basically, so far it has provided
    [1] a "rough draft" (published a few years ago in Science and Nature) of the base sequences of all 30,000 human genes
    [2] and maps of where these genes are located on chromosomes, 
    but they don't yet know what all of these genes do-- for many of the genes they don't know which ones are for which proteins or what effect they have on phenotypic traits or where some disease alleles are.  And there are questions about accuracy and possible overlap of some genes sequenced by more than one lab.  
  • However, the HGP is now at the point where the information it has accumulated can begin to be applied to solving real problems about specific genetic diseases (see textbook specifics below). 
  • For finding the gene when the HGP hasn't figured out which one it is, researchers typically work backwards, starting with the mRNA identified as being unique to diseased tissue or a specific functional protein, then creating cDNA fragments which can be analyzed by hybridizing parts of them with a series of probes.  See http://vector.cshl.org/dnaftb/39/concept/index.html for a more coherent explanation and the  general idea of how they made the HGP move faster than expected.  See http://vector.cshl.org/Shockwave/cycseq.html for an explanation of how they sequenced the parts which didn't hybridize with probes which already had their sequencing done.  
• Main Textbook points.  The most important parts are on pp. 324-332, especially the summary and Activity 16.1 Human Genome Sequencing Strategies.
• Sequencing:  
  • Review tool #6
  • Do CD Activity 16.1 Human Genome Sequencing Strategies and/or study pages 318-321, especially figures 16.2, 16.3, 16.4, and 16.5.  Make sure you understand
    •  BACs (Creating a Map) and plasmids
    • clones, cDNA, and libraries (explained better on CD activity 17.1   Creating a cDNA Library (good review of reverse transcriptase, cDNA, restriction endonuclease, complementary base pairing, ligase, plasmid, library)  
    • shotgun sequence
    • ORFs
    • easy explanation of the dog genome technique http://www.genomenewsnetwork.org/articles/09_03/dog_genome.shtml 
• GENOMES.  
  • Prokaryotic genomes and lateral transfer.   Much of this information is still speculative but scan pp. 321d-324 and figure out why lateral transfer is an important concept.
  • Eukaryotic genomes, pp. 324-332.  Overall, focus on these points:
    • What practical uses do we expect from the genome sequences of human beings?
    • Why do we care about the genomes of non-human critters?
    • Just a year ago, scientists figured out that human beings have only 30 thousand genes which are actually transcribed.  The vast majority of our DNA is "non-coding."  
      • How do transposable elements (fig. 16.7) transpose themselves?  Why?
      • How did hypervariable micro- and mini-satellites originate?  Why are they more variable than coding genes?  How could you make a DNA fingerprint, and why would you choose noncoding DNA?  Describe some practical uses for DNA fingerprints.
    • How could you make a gene chip or a proteonomic chip?  Why?  
    • Will genome research have medical implications?  Give some examples
    • Note the privacy essay, which may come in handy in your report for Lab 11.

• Checklist:  genomics, whole-genome sequencing, Human Genome Project, dideoxy  sequencing, dideoxyribonucleotides, electrophoresis, autoradiography, gel-filled capillary tubes, fluorescent markers, bacterial artificial chromosome (BAC), BAC library, DNA microarray (gene chip), proteomics, rational drug design, kilobase (kb), 160-kb fragment, physical map of a chromosome, map-based sequencing, shotgun sequencing, repeated sequences, introns, raw sequence data, open reading frame (ORF), reverse transcriptase, complementary DNA (cDNA), cDNA library, bioinformatics, redundancy, plasmids, lateral gene transfer, G-C to A-T composition, comparative genomics, E. coli O157:H7, Shigella, dysentery, transposable elements, reverse transcriptase, DNA transposon, transposase, repeats, microsatellites and minisatellites, hypervariable sequence, DNA fingerprinting, polymerase chain reaction (PCR), gene family, pseudogene, polyploid, alternative splicing hypothesis, functional genomics, 
• Preview of Quiz and Test Questions
  • Chapter 16.1  Content Review #1, 2, 3, 4; Conceptual Review #4; Applying #3, 4

• Study CAREFULLY the introduction and pituitary dwarfism case study. especially fig. 17.1 and 17.2 and CD activity 17.1    Finding the Growth Hormone Gene, pp. 335-339a.  Be prepared to discuss this case in detail.
• Be able to explain how we find the genes which cause a particular disease.  Be sure to explain the roles of pedigrees, markers, RFLPS, and "fingerprint-type" banding patterns  on pp. 339-344a, especially figs. 17.3, 17.4b, 17.5a and 17.5b, 17.6a and 17.6b, and 17.7.  
• See box 17.1 and 17.2:  how can we screen individual people to see if they have the "bad" allele?  Also check out  http://genomics.phrma.org/medicine.html 
  • RFLPs are rough indicators (and sometimes RFLPs are markers or base sequences which are often inherited with the allele because they are linked closely on the same chromosome).
  • SNPs are exact base sequence differences which pinpoint the molecular difference between the two alleles.  SNPs, or single nucleotide polymorphisms, are variations involving only one base in a DNA sequence, or a gene. SNPs are different from RFLPs (which often have differences in many bases and are useful in identifying suspects, etc. but may not correspond to any known change in a phenotype). SNPs can be used to detect actual genes, including some alleles directly related to diseases. One issue involving SNPs is whether SNP data should be available to all researchers or whether commercial laboratories can patent SNPs so as make money (for example, by developing genetic screening tests). For more information, check the SNP database or go to the library to find this reference: Science 277:1752 or search other locations for "SNPs."
  • but for both you need a probe and a blot or gene chip to show that the RFLP or SNP is there.
• Gene therapy is a topic you need to be prepared to discuss in detail:  pp. 344-347a.     Keep this in mind for Lab 8.  Also check news:             
                 http://www.phrma.org/genomics/today/index.htm.
• What causes mutations which cause genetic disease?  Review mutations, from chapter 12
• How will the Human Genome Project help with any of the above? Think again about chapter 16.

• Checklist:  biotechnology, genetic engineering, recombinant DNA technology, restriction enzymes, sticky ends, complementary base pairing, DNA ligase, recombined sequences, "designer genes," pituitary dwarfism, pituitary gland, growth hormone, GH1, GH1 protein, recessive trait, prions, Creutzfeldt-Jakob disease, recombinant E. coli cells, reverse transcriptase, DNA polymerase, complementary DNA (cDNA), cDNA library, plasmids, antibiotic, antibiotic resistance, vector, transformation, probe, nucleic acid hybridization, pedigree, sex chromosome, sex-linked traits, hemophilia, X-linked recessive allele, carriers, autosome, autosomal recessive disease, autosomal dominant disease, genetic markers, coinheritance, restriction enzyme recognition sites (palindromic), single nucleotide polymorphisms (SNPs), Huntington's disease, huntingtin, apoptosis (2002 Nobel Prize), transgenic mice, animal model, carrier testing, prenatal testing, amniocentesis, chorionic villi testing, adult testing, gene therapy, retroviruses, adenoviruses, severe combined immune deficiency  
• PREVIEW of QUIZ AND TEST QUESTIONS 
  http://wps.prenhall.com/esm_freeman_biosci_1/0,6452,499245-,00.html
  • p. 350 Content:  #1, 2, 3, 4, 5, 6; Conceptual #1,2,3,4,6; Applying #1,3 (for Lab 11), Figure review #1 & 2

USING THE TOOLS for Agriculture and for testing hypotheses

• Only a few textbook pages to study:  347-350.  Then we'll review the lessons above.
• Checklist:  transgenic, Bacillus thuringiensis, Bt toxin, glyphosate, vitamin A, rice endosperm, crown gall, Ti plasmid, T-DNA, virulence genes, gene gun
• review Expressing a Gene in Bacteria (recombinant, promotor, plasmid)  
• PREVIEW of QUIZ AND TEST QUESTIONS
  • http://wps.prenhall.com/esm_freeman_biosci_1/0,6452,499245-,00.htmlp. 351 Conceptual #5; Applying # 2 (for Lab 8)

  • which tools are used (and how and why) for each of these:

    1. Does this DNA "fingerprint" match the suspect's?  Is his father really his father?  Were these babies switched at birth?  What does forensic mean?     
    2. Can we change the bad alleles?  How do we know that bad alleles are really bad?
    3. Has inbreeding eliminated genetic biodiversity in this population?  
    4. Did these two species share the same ancestor?   
  • MORE

    1. A technique for making many copies of a DNA segment is called 
       [a] blotting. [b]electrophoresis. [c] hybridization. [d] PCR. [e] recombination.

    2. A critter which contains genes which came from another species is called  
       [a] a hybrid.   [b] a mutant.  [c] a freshman.  [d] a vector.   [e] recombinant.   [f] transgenic.

    3. A plasmid is an example of      
       [a] a hybrid.   [b] a mutant.  [c] a freshman.  [d] a vector. 
       [e] recombinant DNA.   [f] transgenic DNA.

    4.  DNA fingerprint is a photograph of fragments separated by a process called
       [a] blotting. [b] electrophoresis. [c] hybridization. [d] PCR. [e] recombination.

    5. The fragments in a DNA fingerprint are called
       [a] RFLPs.   [b] SNPs.     

    6. You can "see" the fragments because they're hybridized with radioactive
       [a] blots.   [b] enzymes.  [c] primers.  [d] probes.
    7. The human genome project involves mapping the loci of genes and also
       [a] finding cures for genetic diseases. [b]  finding the base sequence of genes.
       [c] finding the base sequence of proteins. [d] both "a" and "b."     [e] both "b" and "c."

    8. A gene inserted into a plasmid is an example of      
       [a] a hybrid.   [b] a mutant.  [c] a freshman.  [d] a vector. 
       [e] recombinant DNA.   [f] transgenic DNA.         you want answers?
       

  • SAMPLE "ESSAY" QUESTIONS from old news (new news essays may be added soon):
    • Read this science news article- http://www.nature.com/nsu/020923/020923-11.html What is the hypothesis?  What evidence supports it?   What are some implications or applications of these experiments beyond knowing more about these beetles?  
    • Try:  http://www.eurekalert.org/pub_releases/2003-09/uomh-ngf092503.php.  How is the disease inherited?  How did the researchers find out?  What is wrong with the "bad" allele?  Why would this error be a problem?    
    • How about the dog genome:  http://www.eurekalert.org/pub_releases/2003-09/tifg-dgp092303.php.  or http://www.genomenewsnetwork.org/articles/09_03/dog_genome.shtml Why and how did they do it?  What was especially interesting?  
    • Read this one: http://health.ucsd.edu/news/2003/09_28_Hamilton.html What is a retrovirus?  What surprised the researchers for this one? "...sophisticated laboratory techniques called positional complementation to identify Mvb1...":  what do you think positional complementation is most like (of the tools you have studied?) 
    • Try http://www.hopkinsmedicine.org/press/2002/September/020925B.htm.    What is a complex disease?  What are some other examples of complex diseases?  What is a marker?  Why did they use markers instead of probes with SNPs?  
    • For any "new" news from News stories or from http://science.bio.org/  at the very least be prepared to
      • Write an "experiment summary form for any experiments described
      • Explain any  "tools" described.
      • Explain why this story was in the news and how it relates to stuff you're supposed to know.

     

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    TAKE-HOME QUIZ due Wednesday, 15 Oct Print out a recent gene news article found at    http://science.bio.org/   (Don't use one we discussed in class) Turn it in with either a] six technical gene terms circled in the text         with your definitions attached or b] An experiment summary form Do both or do two articles if you need an extra quiz to replace ANY old quiz.

     

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LINKS for more information