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Bio 201 Lessons 8-9

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  • Production of multiple copies of a specific DNA segment

    DNA Cloning

  • Enzyme that seals the bond on the sugar phosphate backbone; joins the Okazaki fragments to form a continuous DNA strand

    DNA Ligase

  • Small, circular double-stranded DNA molecule that replicates separately from the genome

    Plasmid

  • Where were plasmids originally found?

    In bacteria

  • What do plasmids hold?

    A small number of genes

  • Plasmid that has been genetically engineered for efficient DNA cloning

    Vector

  • Endonucleases that cut at specific nucleotide sequences

    Restriction Enzymes (Restriction Sites)

  • Sequences that have the same 5' to 3' on each strand

    Palindromic Sequence

  • Process in which the double stranded recombinant DNA molecule is introduced into bacterial cells

    Transformation

  • Technique used to isolate plasmid DNA

    Mini-Prep

  • Technique used to separate and visualize DNA fragments

    Gel Electrophoresis

  • True or False: Smaller DNA fragments will migrate faster down in gel electrophoresis

    True

  • What charge are the fragments that move down towards the positive electrode in the gel matrix?

    Negative

  • Why can we not just place a human gene into bacteria?

    Bacteria don't like linear DNA and cannot replicate it.

  • Why do we need a cloning vector?

    A vector (plasmid) is small, easily manipulated, and can be easily copied

  • What are the three components of a cloning vector?

    1)Multiple Cloning Site

    2)Selectable Marker

    3) Origin of Replication

  • Why is the origin of replication important?

    Its necessary for bacteria to copy the DNA

  • True or False: A multiple cloning site (MCS) will have lots of restriction sites to choose from that only occur once in the vector

    True

  • What criteria must a restriction enzyme meet so that a gene can be cloned into the multiple cloning site?

    1) Must be on both sides of the gene without cutting inside of it

    2) Must be in the multiple cloning site

  • What does it mean for sticky ends to be compatible with each other?

    The gene and vector must be cut with the same enzyme

  • Why do sticky ends contribute to the efficiency of a restriction enzyme's ligation?

    The sticky bonds can temporarily hold pieces together until ligase forms the phosphodiester bond

  • True or False: A gene can make lots of copies because the plasmid does not have an origin of replication

    False, a plasmid with an origin or replication will produce a lot of DNA copies

  • Selection of cells that contain a certain vector or gene

    Positive Gene Selection

  • What enzyme does the LacZ gene produce if it is intact?

    B-galactosidase

  • What is the substrate of the enzyme B-galactosidase? What color does this substrate produce?

    X-gal, blue

  • What additional steps happen in human cells that might NOT happen in bacteria?

    RNA Processing & Secretion

  • What toolbox technique allows us to confirm the presence of a specific protein?

    Western Blot & ELISA

  • Enzyme that creates new DNA molecules by assembling individual nucleotides

    DNA Polymerase

  • Enzyme that breaks weak hydrogen bonds between complementary base pairs in DNA or RNA

    Helicase

  • Enzyme that breaks phosphodiester bonds in the backbone to relieve tension far ahead of replication machinery

    Topoisomerase

  • Enzyme that repairs the bonds in the DNA backbone

    Ligase

  • What does it mean for replication to be semi-conservative?

    Each parent strand serves as a template for the new molecule

  • What strands are present in a new molecule after replication?

    1 Parent Strand & 1 New Strand

  • True or False: DNA polymerase is able to add nucleotides to the 5' end of a growing DNA strand

    False, DNA polymerase always adds to the 3' OH

  • Why is there RNA during DNA replication?

    Primase first adds RNA primer at the replication bubble so that DNA polymerase has a 3' OH to add to

  • True or False: DNA polymerase can't start from nothing

    True

  • Where does DNA polymerase move on both strands?

    Away from the replication bubble

  • What results in the formation of the lagging strand?

    Antiparallel nature of two DNA strands

  • Where are the most recently Okazaki fragments located from the RNA primer?

    The farthest away from the RNA primer and closest to the replication forks

  • Enzyme that removes the RNA primer at the beginning of each Okazaki fragment and fills in the gap

    DNA Polymerase I

  • What features are unique to eukaryotic DNA replication?

    Linear chromosomes and many origins of replication

  • What features are unique to bacterial DNA replication?

    Circular chromosomes and one origin of replication

  • What does it mean for components to be conserved across bacteria and eukaryotes?

    Evidence of common ancestry (evolution)

  • What would happen to DNA if topoisomerase were inhibited?

    DNA would become tightly coiled as replication began and the replication process could not proceed as DNA would not be open to DNA polymerase

  • Method used to fluorescently label specific DNA sequences

    Fluorescent in Situ Hybridization (FISH)

  • What happens during normal replication but there is no telomerase present?

    The lagging strand gets shorter with each round of replication. Both ends of the lagging strand get shorter

  • Process by which a cell ages and permanently stops dividing but does not die

    Senescence

  • Enzyme that adds nucleotides to the telomeres

    Telomerase

  • Why are cancer cells able to keep dividing?

    The telomeres express telomerase resulting in the cells not senescing

  • Technique used to amplify (make many copies) of a specific stretch of DNA

    PCR

  • True or False: We can use any cells of the body because they all have the same DNA in their nucleus

    True

  • What are the 4 components necessary for PCR?

    1) Genomic DNA containing the target gene

    2) All four DNA nucleotides

    3) Heat stable DNA polymerase

    4) DNA primers (two different sequences)

  • What are the 3 steps that happen in a cycle of PCR?

    1) Hydrogen bonds disrupted between two strands of DNA (Denaturation)

    2) DNA primers anneal to target regions (Annealing)

    3) Taq polymerase adds free nucleotides to synthesize a new, complementary strand of DNA (Extension)

  • What makes taq polymerase better for PCR than other enzymes?

    It has a stable protein structure due to its tightly packed hydrophobic regions

  • True or False: Primers facing away from the gene will amplify the whole segment of DNA

    False, if a primer is facing away from the gene, the polymerase would be adding to the 3' end

  • In Vivo or PCR: Heat separates the two strands of DNA

    PCR

  • In Vivo or PCR: Helicase separates the two strands of DNA

    In Vivo

  • In Vivo or PCR: Taq polymerase is the enzyme that elongates new strand of DNA

    PCR

  • In Vivo or PCR: DNA polymerase is the enzyme that elongates new strand of DNA

    In Vivo

  • In Vivo or PCR: Primers made of DNA

    PCR

  • In Vivo or PCR: Primers made of RNA

    In Vivo

  • Is ligase needed in PCR?

    No because there is no lagging strand

  • Is ligase needed In Vivo?

    Yes because the Okazaki fragments link onto the lagging strand

  • What makes PCR and In Vivo similar?

    Nucleotides are needed for elongation, DNA serves as a template for new strand, and 5' --> 3'

  • What are the 4 components required by both eukaryotes and prokaryotes for DNA replication

    1) Double Stranded DNA

    2) Four types of DNA nucleotides

    3) Primers

    4) Origins of Replication

  • What does DNA polymerase require in order to synthesize a complete strand of DNA?

    1) 3' OH end of the new DNA strand

    2) Single-stranded DNA template

    3) All four types of nucleotides

  • What happens to cells that fail to take up a plasmid on ampicillin/X-gal plates?

    They will die because they did not transform

  • What molecular processes (steps in central dogma) are involved in genetic selection?

    Replication, transcription, translation, and protein function

  • What direction does helicase move in?

    The same direction as the replication fork

  • What enzymes are required for in vivo DNA replication, but not for PCR?

    Helicase, Primase, Ligase, and Topoisomerase