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shockwave
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BIOTEC E2 C3
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2011-10-18 18:17:56
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BIOTEC E2 C3
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BIOTEC E2 C3
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  1. Probes are made using ----- ------------.
    DNA synthesizers.
  2. Can use DNA, RNA or protein sequence to design probes and create ----------.
    Oligonucleotides
  3. Degenerate oligonucleotides must be made when using ---- ----- ----- to make a probe.
    amino acid code.

    Remember several codons can give the same amino acids.

    Want all possibilities taken into account
  4. BASICALLY, HOW DO YOU MAKE AN EXPRESSION LIBRARY?
    Made with a cloning vector containing the required regulatory elements for gene expression.

    Promoter regions and operators.
  5. In E.coli expression vector, the promoter is placed next to WHAT?
    a unique restriction site
  6. When foreign DNA is cloned into an expression vector in the ------- ------- ----the gene is transcribed and translated in E.coli
    proper reading frame
  7. Libraries created this way only allow cDNAs inserted in frame to be expressed.
    When foreign DNA is cloned into an expression vector in the proper reading frame the gene is transcribed and translated in E.coli.

    Useful in identifying clones that make a protein where an antibody is available
  8. T OR F?
    Immune responses in eukaryotes produce specific antibodies to foreign proteins
    TRUE
  9. T OR F?
    Antibodies can be used in place of nucleic acid probes to identify the clone containing the gene expressing the specific protein
    TRUE
  10. WHAT THE HELL AM I?
    Is connected to the gene of interest and used to indicate if the desired DNA is expressed. (INDIRECTLY).
    Allows researchers to measure how well a gene is expressed.
    REPORTER GENES
  11. 3 EXAMPLES OF REPORTER GENES
    • Luciferase gene
    • Green fluorescent protein (GFP)
    • β-glucuronidase gene (GUS)
  12. Found in firefly and the bacteria Vibrio harveyi, this gene produces light in response to the molecules luciferin and ATP. ALDEHYLDE IS ALSO PART OF THIS REACTION.
    Luciferase gene
  13. Produced by the jellyfish Aequorea victoria and interacts with the protein aequorin to produce fluorescence. Needs strong promoter because --- expression is generally weak. The --- gene can be fused with another gene, allowing --- to indicate the production of the desired protein.
    Green fluorescent protein (GFP)
  14. Encodes an enzyme that breaks down chemicals called β-D-glucuronides. Can produce a blue or fluorescent color.
    β-glucuronidase gene (GUS)
  15. Method used to locate specific regions within cloned genes of DNA inserts.

    Done by cutting large inserts with restriction enzymes and separated on an agarose gel (can’t probe a gel easily).
    • SOUTHERN BLOT HYBRIDIZATION.
    • -HYBRIDIZED DNA FRAGEMENTS.
    • -To identify the fragment that contains your gene of interest probes must be used.
  16. Southern blotting was developed
    WHO & WHEN?
    Edward Southern in the 1970s
  17. NAME THE 7 STEPS OF THE SOUTHERN BLOT HYBRIDIZATION METHOD.
    • 1. DIGEST DNA WITH RESTRICTION ENDONUCLEASE.
    • 2. PERFORM ARAGROSE GEL ELECTROPHORIES ON DNA FRAGMENTS.
    • 3. DNA FRAGMENTS FRACTIONATED BY SIZE.
    • 4. TRANSFER GEL TO NITROCELLULOSE FILTER.
    • 5. MAKES A COPY OF GEL.
    • 6. HYBRIDIZE FILTER WITH RADIOACTIVE LABELED PROBE. (PLACE GEL AND LABEL IN A BAG).
    • 7.EXPOSE FILTER TO X-RAY FILM. RESULT SHOWS HYBRIDIZED DNA FRAGMENTS.
    • 8. CUT THE GEL INTO LITTLE PIECES AND SERVE TO UNWANTED GUESTS. DELICIOUS!
  18. WHAT IS THE SOUTHERN BLOT CAPILLARY TRANSFER?
  19. The ----- is denatured during the processed resulting in single strands that go to the nylon membrane during the blotting technique.
    DNA
  20. WHAT IS USED IN A SOUTHERN BLOT TO VISUALIZE IT?
    AUTORADIOGRAPHY
  21. NAME STEPS 1 AND 2 OF THE SOUTHEN BLOT METHOD.
    • 1. DIGEST DNA WITH RESTRICTION ENDONUCLEASE.
    • 2. DO A GEL ON DNA FRAGMENTS FROM DIFFERENT DIGESTS.
  22. SOUTHERN BLOT METHOD...STEP 3. WHAT IS THE NITROCELLULOSE SOAKED IN?
    ETHIDIUM BROMIDE
  23. WHAT ARE STEPS 3,4 & 5 OF THE SOUTHEN BLOT TEST.
  24. NAME TWO WAYS FOR USING A BLOTTING APPLICATION FOR MEDICAL DIAGNOSTICS.
    Medical diagnostics for identifying a genetically inherited disease.

    1.Defective genes may only have a point mutation, but if it occurs in a restriction site it will be detected using this method.

    2.Generation of variable pattern is useful in detecting normal and variant genes
  25. Southern blotting has many uses,
    NAME 2.
    • 1. Detects differences in banding patterns between organisms or within DNA.
    • 2. Restriction fragment polymorphisms (RFLP)(MEDICAL DIAGNOSTICS CLASSIFIED UNDER THIS).
  26. WHAT THE HELL IS RFLP?
    Restriction fragment polymorphisms (RFLP).

    • Used to generate individual DNA fingerprints.
    • Individuals are identified by inherited genetic traits (maternity, paternity, crime).
  27. WHAT'S THE BASIC DIFFERENCE BETWEEN NORTHEN,SOUTHERN AND WESTERN BLOTTING?
    • NORTHERN IS RNA
    • SOUTHERN IS DNA
    • WESTERN IS PROBING PROTEIN GELS.
  28. WHAT BLOTTING METHOD WOULD ONE USE TO STUDY RNA?
    NORTHERN
  29. Look at pattern of protein produced at a given time.
    Two dimensional gels used to separate proteins of like size and charge.
    Isolate proteins for further structural or biochemical study.
    WHAT BLOTTING METHOD IS THIS?
    WESTERN
  30. WHAT BLOTTING METHOD HAS Two dimensional gels used to separate proteins of like size and charge?
    WESTERN
  31. IN THE WESTERN BLOTTING EXAMPLE FIG 4.26 WHAT TYPE OF MEDIA WS USED FOR THE GEL AND WHY?
    • POLYACRYLAMIDE.
    • SIZE OF THE HIV PROTEIN.
  32. IN A WESTERN BLOT, HOW MANY ANTIBODIES ARE USED?
    • TWO.
  33. T OR F?
    DNA PROBES DON'T USUALLY USE 2 ENZYMES, BUT ANTIBODIES USUALLY DO.
    TRUE.
  34. -----uses components of DNA replication to copy DNA in a tube: Can rapidly isolate a specific DNA sequence without screening a library.
    Polymerase Chain Reaction (PCR).
  35. Selectively amplify sequences without hybridization.
    WHAT METHOD?
    PCR
  36. T OR F?

    Utilizing selective amplification, Any DNA can be isolated from the total DNA of an organism or a community.
    • TRUE.
    • THINK PCR.
  37. T OR F?
    IN PCR the sequence of the flanking regions of the gene to be amplified must be known.
    TRUE
  38. IN PCR YOU REPLACE THEN ENZYMATIC PROCESS OF DNA REPLICATION WITH WHAT?
    HEAT.
  39. HOW MANY PRIMERS DOES ONE USUALLY NEED TO DO PCR?
    • TWO.
    • ONE FORWARD (3 TO 5)
    • ONE REVERSE (5 TO 3).


    Two short oligonucleotide primers are used to start the amplification process during each round of PCR
  40. Other than primers and sample DNA, several reaction components are needed.
    NAME 3
    • 1.Taq DNA polymerase (Thermus aquaticus) Stable in temps needed to denature DNA. (LIKES IT HOT).
    • 2.All four deoxyribonucleotides.
    • 3.Buffer with proper magnesium concentration.
  41. WHAT IS THE SOURCE OF Taq DNA polymerase ?
    WHY IS IT USED?
    • Thermus aquaticus.
    • STABLE IN HIGH TEMPS THAT WOULD OTHERWISE DENATURE DNA.
  42. SHORT FRAGMENTS OF DNA CALLED -------- ACT AS PRIMERS TO ALLOW A SPECIFIC DNA TO BE REPLICATED BY DNA ---------.
    • OLIGONUCLEOTIDES
    • POLYMERASE
  43. THE APPROXIMATELY ----- TO --- NUCLEOTIDE PRIMER BASE PAIRS WITH THE TEMPLATE DNA TO INITIATE REPLICATION IN THE TEST TUBE.
    18-21
  44. NAME 3 THINGS THAT ARE OCCUR DURNING THE AMPLIFICATION CYCLE OF PCR.
    1.Template is denatured by high temperature.
  45. 2.Primers are annealed by lowering the temperature.
  46. 3.DNA polymerase extends the DNA from the primers.
  47. IN PCR Repeated cycles of denaturation, primer annealing, and DNA synthesis result in ------- ---------.
    exponential amplification
  48. IN THE PCR AMPLIFICATION CYCLE, Normally --- TO ---- cycles are used in a thermal cycler
    • 25-40.
    • adverage about 30...per gsell in lecture.
  49. The thermal cycler starts at 95 then drops to 55 and then to 72.
    WHY STOP AT 72?
    • Taq DNA polymerase
    • (Thermus aquaticus)
    • PERFERS 72.
  50. THE PCR IS AN ------ ------- DNA REPLICATION METHOD
    IN VITRO

    Latin: within glass
  51. WHAT ARE THE FIRST 4 STEPS OF PCR?
  52. WHAT ARE THE LAST 3 STEPS IN PCR?
  53. BECAUSE OF IT'S VERSATILITY, PCR CAN BE USED AT LEAST 4 DIFFERENT WAYS. NAME THEM.
    1.Rapidly isolate specific sequence for cloning.

    2.Identify specific genetic loci; diagnostic/med.

    • 3.Generate DNA fingerprints (forensics)
    • 4. Rapidly sequence DNA.
  54. DNA Sequencing methods are valuable, NAME THE FIRST 4 REASONS WHY/HOW.
    1. Methods are used extensively for research like the Human Genome Project, evolution studies, mutation detection.

    2.Confirm identity of library genes isolated by PCR or hybridization.

    3. Determine DNA sequence of promoters and other DNA regulatory elements controlling expression.

    4.Reveal the fine structure of genes and other fragments of DNA.
  55. T OR F?
    SEQUENCING DNA HELP Confirm DNA sequence of cDNAs and other PCR amplified products.
    TRUE
  56. T OR F?
    DNA SEQUENCING HelpS deduce the amino acid sequence of a gene or cDNA sequence
    TRUE
  57. T OR F?
    COMPUTERS ARE USED FOR DNA SEQUENCING?
    • TRUE.
    • NO SHIT!
  58. NAME 2 SEQUENCING METHODS AND WHEN?
    • 1977
    • 1. Maxam and Gilbert method (Harvard)
    • Uses selective degradation of bases
    • (A, T, C, G)

    2.Sanger Method (CAMBRIDGE)uses DNA synthesis
  59. DEFINE Maxam and Gilbert SEQUENCE method
    • 1.Uses selective degradation of bases (A, T, C, G)
    • 2. For example C tube has a chemical that removes cytosine and the DNA is then cut where the missing cytosine was.
    • 3. When run together on a gel (radiolabled 5’) DNA patterns for all 4 nucleotides fall into a pattern that can be read by eye, or computer.
  60. THIS SEQUENCING Method uses dideoxynuclotides for all 4 bases.
    SANGER
  61. DEFINE SANGER SEQUENCING METHOD
    • 1. Involves the termination of newly synthesized DNA strands at specific sites.
    • -DNA primer is annealed to the denatured template DNA, polymerase extends the sequence from the primer (radiolabeled).

    • 2. Method uses dideoxynuclotides for all 4 bases.
    • -These are added in at random points and terminate DNA synthesis.
    • -These molecules lack a 3’ hydroxyl.
  62. The key principle of the Sanger method was the use of ----------- ------- as DNA chain terminators
    dideoxynucleotide triphosphates (ddNTPs)
  63. HOW DO YOU SET UP A SANGER SEQUENCE?
    • 1.a single-stranded DNA template.
    • 2.DNA primer.
    • 3. DNA polymerase,
    • 4. normal deoxynucleotidetriphosphates(dNTPs)
    • 5. Modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation.
  64. WHAT GOES INTO THE 4 TUBES FOR A SANGER SEQUENCE?
    • 1. all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP).
    • 2. DNA polymerase.
    • 3.To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP)
  65. WHAT IS THE FUNCTION OF THE dideoxynucleotides?
    (ddATP, ddGTP, ddCTP, or ddTTP)
    The chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.
  66. WHERE IN THE HELL IS THE 3' END WHERE THE -OH GROUP SHOULD GO ON A ddATP dideoxynucleotide?
  67. WHAT IS THE END RESULT WHEN YOU RUN A GEL AFTER SEQUENCING?
    HAVE THE ABILITY TO SEE THE LENGTH OF EACH OF THE CHAINS THAT WERE PRODUCED. FROM SHORTED TO LONGEST AND THE AMOUNT OF BASES IN A CHAIN.
  68. PROTEIN RESEARCH FOCUSES ON WHAT?
    NAME 4.
    1. Amino acid sequence (from DNA).

    2. Chemical properties of protein.

    3. Predicted 3-D structure (pure protein/computer analysis).

    4.Properties of proteins are modified by altering the amino acids in the protein.
  69. --------------- ------------ mutagenesis introduces desired base change into the target DNA during replication
    Oligonucleotide-directed
  70. WHAT ARE THE 4 STEPS IN
    Oligonucleotide-directed mutagenesis?
  71. NAME 3 TYPES OF NATURAL IMMUNITY
    • PHAGOCYTES
    • PHYSICAL
    • CHEMICAL
  72. NAME 2 TYPES OF ACQUIRED IMMUNITY
    CELL AND ANTIBODY MEDIATED.
  73. DEFINE ANTIBODIES
    Immunoglobulins (Igs).

    Proteins that protect an organism by binding to and eliminating foreign molecules (antigens)
  74. T OR F?
    ANTIBODIES ARE USED FOR THE DETECTION OF GENETIC AND AQUIRED DISEASE.
    TRUE
  75. WHAT THE HELL IS AN APITOPE?
    • AN ANTIGENIC DETERMINANT.
  76. AN ANTIBODY IS COMPOSED OF ----- POLYPEPTIDE CHAINS JOINED IN THE SHAPE OF A ------.
    • 4, Y
  77. Antigen binding site varies, determined by -------- ------- OR -------- ---- from both light and heavy chain
    • amino terminus OR
    • hypervariable region (H2L2)
  78. Produced by bone marrow and cirulate in blood and lymph.
    B lymphocytes which produce immunoglobulins
  79. Antigen specificity is determined by WHAT?
    Ig that a B cell synthesizes; proliferates only in Ag presence.
  80. NAME THE 5 Ig CLASSES
    G A M E D
  81. WHAT THE HELL DOES AN IgA LOOK LIKE?
  82. WHAT 3 Ig HAVE THE SAME SHAPE?
  83. WHAT THE HELL DOES AN IgM LOOK LIKE?
  84. T OR F?
    B cells proliferate in response to specific antigen (T-Cell helps). That same specific antibody (monoclonal) produced by progeny B plasma cells.
    TRUE
  85. DEFINE hybridoma
    a hybrid cell formed by the fusion of a myeloma cell and an antibody-producing cell. Hybridomas are used to produce monoclonal antibodies.
  86. HOW DOES ONE PRODUCE MONOCLONAL ANTIBODIES?
    By fusing a B-cell with immortal cancer cells (hybridoma)
  87. NAME 4 APPLICATIONS FOR MONOCLONAL ANTIBODIES.
    1.Identify specific proteins on a gel.

    2.Identify specific clones from an expression library.

    • 3.Medical diagnostics (Immunoassays); for hormones, and antigens (blood group, pathogens, or disease products.
    • 4.Prevent graft vs host rejection to remove T cell from donor and prevent attack.
  88. This technique detects antigens in cells or tissues that are bound to a microscope slide.
    • Fluorescent Antibody Technique
    • Also called immunofluorescence microscopy
  89. WHAT IS THE MOST COMMON TAG IN FAT (Fluorescent Antibody Technique)?
    fluorescein isothiocyanate (FITC)
  90. NAME THE 2 TYPES OF Fluorescent Antibody Techniques
    DIRECT AND INDIRECT ASSAY.
  91. DEFINE DIRECT ASSAY OF THE Fluorescent Antibody Technique.
    • Fluorescent antibodies are attached directly to the cell surface. This way examines tissues for infection.
  92. WHAT Fluorescent Antibody Technique would one use to exam tissue for infection?.Direct or indirect?
    DIRECT ASSAY.

  93. Define indirect assay of the Fluorescent Antibody Technique.
    • Unlabeled antibodies are first applied, followed by a secondary fluorescent antibody specific to the unlabeled antibody. Can detect whether antibodies are produced in response to an infection.
  94. What immunofluorescence microscopy assay can detect whether antibodies are produced in response to an infection.
    Direct or indirect?
    INDIRECT ASSAY.

  95. Detects specific proteins, usually antibodies, in serum, and is extremely specific.
    • Enzyme-Linked Immunosorbent Assay
    • (ELISA)
  96. The label for the antibody is an enzyme that can catalyze a chemical reaction.WHAT ASSAY?
    Enzyme-Linked Immunosorbent Assay. (ELISA)
  97. The steps of an ELISA are:
    (5 OF THEM)
    1.Bind the antigen to a plastic microplate.

    2.Wash the wells of the microplate, and add serum sample, so antibodies can react to the antigen if they are made.

    3.Wash wells, and add a secondary antibody specific for the antibody in the serum to the plate. This antibody is labeled with the enzyme.

    4.Wash the wells and treat the plate with the enzyme’s substrate.

    5.A color change is a positive result, which can be quantified based on the intensity of color developed.
  98. WHAT THE HELL IS THIS?
    ELISA
  99. T OR F ?
    oligonucleotides can be used for PCR or hybridization for gene isolation.
    TRUE
  100. DEFINE Edman Degradation.
    • AMINO ACID ID PROCESS.
    • First amino acid is chemically modified to phenylthiocarbamoyl amino acid and removed by acid treatment for identification (GC). Repeated until polypeptide sequence is identified.
  101. WHAT THE HELL IS THIS?
    EDMAN DEGRADATION.
  102. Phenylthiocarbamoyl amino acid.
    WHAT METHOD?
    EDMAN DEGRADATION
  103. Very similar to Southern blot hybridization (x1000s), and can be used to analyze gene activity for thousands of genes on one array.
    DNA MICROARRAY TECHNOLOGY
  104. Used to study gene expression (transcription) in different conditions, cells or stages of development.
    WHAT ARRAY?
    DNA MICROARRAY TECHNOLOGY
  105. With this array, one can follow changes in genomic DNA and detect mutations down to single base changes
    DNA MICROARRAY
  106. WITH THIS ARRAY, ONE CAN COMPARE GENE PATTERNS OF NORMAL VS CANCER AND STUDY THE EFFECTS OF TREATMENTS AND DRUGS.
    DNA MICROARRAY
  107. DEFINE "in situ"
    to examine the phenomenon exactly in place where it occurs (i.e. without moving it to some special medium).
  108. WHAT THE HELL DOES "IN VIVO" MEAN?
    Latin for "within the living" is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro ("within the glass", i.e., in a test tube or petri dish) controlled environment.
  109. NAME THE 2 DYES IN A MICROARRAY.
    • Cy3 GREEN DISEASED STATE
    • Cy5 RED NORMAL STATE
  110. DEFINE THE MICROARRAY PROCESS. ALL 7 OF THEM.
    1.Isolating DNA clones, or prepare oligonucleotides for the microarray (probes). Can be genomic or cDNA sequences or clones.

    2.Apply cDNA or oligonucleotides to a substrate or chip.

    3.Isolation of total RNA or mRNA from a cell or a tissue.

    4.Preparation of cDNA by reverse transcription.

    5.Labeling of DNA with the fluorescent dyes Cy3 (green), for a disease state, or Cy5 (red) for the normal state.

    6.Hybridization of the fluorescently labeled target sample to the microarray and colors are based on ratio of red to green.

    7. Data acquisition and analysis, using lasers and a computer to determine in which states the gene is being transcribed. The fluorescence is converted to numbers (Table 3.3)
  111. NAME THE 3 PRIMARY STEP IN DOING A MICROARRAY.
    THE ARRAY FABRICATION AND THE TARGET PREPARATION. THEN THE IMAGE ACQUISTION AND ANALYSIS.
  112. WHAT THE HELL IS THIS?

    THE ARRAY FABRICATION OF A DNA MICROARRAY.
  113. WHAT THE HELL IS THIS?

    • TARGET PREPRARTION.
    • PREPARING AND HYBRIDIZING LABEL TARGET cDNA.
  114. WHAT IS THIS FROM?

    THIS IS STEP 3 OF 3 OF A DNA MICROARRAY. ANALYZING THE DATA FROM LABELED TARGET cDNA HYBRIDIZED TO A MICROARRAY.
  115. WHAT IN THE GOOD NAME OF THE LORD DOES siRNA MEAN?
    DOUBLE STRANDED RNA ARE PROCESSES INTO APPROX. 21-23 NUCLEOTIDE RNA WITH 2 NUCLEOTIDE OVERHANGS ON THE 3' END.
  116. WHEN WAS RNA interference (RNAi) DISCOVERED?
    1998 CARNEGIE INSTITUTE, MD.
  117. WHAT IS THE RNA interference (RNAi) METHOD? 5 STEPS.
    1.Double-stranded RNAs are processed into small double-stranded RNAs with overhangs on the 3’ end.
  118. 2. RNase enzyme called “Dicer” cuts the dsRNA into short siRNAs.
  119. 3. Each siRNA combines with nucleases to form a RNA-induced silencing complex (RISC).
  120. 4. siRNA unwinds and RISC is activated
    5. Activated RISC targets complimentary mRNA molecules. siRNA strands act as guides where RISC cuts transcripts in an area where siRNA binds, destroying the mRNA
  121. WHAT PROCESS CONTAINS RISC?
    RNA interference (RNAi):

    Each siRNA combines with nucleases to form a RNA-induced silencing complex (RISC).
  122. NAME THE PROCEDURE:

    RNA interference (RNAi).
  123. This method may be a useful method to reduce transcription, reducing gene expression
    • RNAi
    • (RNA INTERFERENCE.)
  124. Recombinant DNA technology can be used in many ways. NAME 5
    • 1.Basic biology
    • 2.Medicine
    • 3.Industry
    • 4.Agriculture
    • 5.Criminal investigation