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BIOMG 3300 > Prelim 2 > Flashcards

Prelim 2 Flashcards

1
Q

Generally speaking, what is the structure and function of DNA?

A

A long thin organic polymer in which the linear sequence of covalently linked nucleotide subunits encodes the genetic message

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2
Q

Given the structures of the four common bases, identify each as a purine or pyrimidine (or describe their general structures)

A

Heterocyclic compounds, aromatic character:
Purines - BIG. two nitro-carbon rings, one with 6C and the other 5C. Connected to pentose by nitrogen at “bottom” of 5C ring.
Pyrimidines - SMALL. one n-c 6C ring, connected to the pentose by the nitrogen at the bottom of the ring.

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3
Q

What are the characteristic components of a nucleotide?

A

1) Nitrogenous base (purine or pyrimidine)
2) pentose
3) one or more phosphate groups

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4
Q

What are the components of a nucleoside?

A

A molecule consisting of a nitrogenous base and a pentose. NO PHOSPHATE.

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5
Q

Convention: How are carbons numbered in the pentose ring of nucleotides?

A

Carbons are designated with ‘ or “# prime” carbon to distinguish them from the base carbons.

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6
Q

Nucleotide structure: Which atom in the sugar is the base attached to?

A

1’ C

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7
Q

Nucleotide structure: What bond connects the sugar and base?

A

N-beta-glycosyl bond.

Formed from removal of H2O. Connects 1’ C to N-1 or N-9 in pyrimidines and purines, respectively.

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8
Q

What is general deoxyribonucleotide structure?

A

Nucleotide (pentose, Pi, & N base). -H group at 2’ C of pentose. Still have 5’ and 3’ -OH available to bind phosphates and other groups.

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9
Q

What is general ribonucleotide structure?

A

Nucleotide (pentose, Pi, & N base). Have -OH groups at 2’, 3’ and 5’ C.

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10
Q

Draw structures representing:

1) a 2’-deoxynucleoside
2) a 2’-deoxynucleotide
3) a 2’-deoxynucleoside 5’-di- or triphosphate

A

1) 2’-Deoxy = no -OH at 2’ C. NucleoSIDE = pentose and base.
2) 2’-Deoxy = no -OH at 2’ C. NucleoTIDE = pentose, base, and one or more Pi groups, unspecified location.
3) 2’-Deoxy = n -OH at 2’ C. NucleoSIDE + 5’-di or triphosphate = 2 or 3 phosphate groups connected at 5’ C.

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11
Q

What characteristic defines DNA vs. RNA?

A

The pentose used in the nucleic bases:
RNA has ribonucleotides.
DNA has deoxyribonucleotides.

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12
Q

What are the purine bases?

A

Guanine and Adenine (GA, big state that makes PURE orange juice).

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13
Q

What are the pyrimidine bases?

A

Cytosine, Thymine, and Uracil. (CT is a small state with a lot of $$$).

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14
Q

What functional group is at the 3’ position in ribo- and deoxyribonucleotides?

A
  • OH
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15
Q

Identify the atom that is found at the 2’ position in deoxyribonucleotides?

A
  • H
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16
Q

In DNA and RNA nucleotides, to which atom in the sugar is the phosphate bound?

A

5’ C

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17
Q

Discuss the nucleotides found in DNA.

A

A, C, T, G. Never U.

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18
Q

Draw a section of DNA (use the letters A, C, G, and T to represent the structure of the base). Point out:

a. phosphodiester linkage
b. sugar-phosphate backbone
c. 5’ and 3’ ends
d. overall charge

A

DO IT.

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19
Q

What bond connects successive nucleotides?

A

Phosphodiester linkages. R-O-(PO2-)-O-R

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20
Q

What is the overall charge of nucleic acid polymers?

A

Negative, because of the negative sugar phosphate backbone.

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21
Q

What gives nucleic ACIDS (RNA, DNA) their acidic character?

A

The negatively charged backbone. The phosphate group acts as an acid much more so than the nitrogenous bases contribute basic character. They are protected on the interior of the duplex, usually.

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22
Q

Convention: What direction are nucleic acid sequences written in?

A

ALWAYS 5’ to 3’.

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23
Q

Distinguish between an oligonucleotide and a polynucleotide.

A

Oligonucleotide is short, or < 50 bases.

Polynucleotides are longer than that.

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24
Q

Discuss the hydrogen bonds that stabilize the DNA double helix.

A

Aromatic and relatively hydrophobic bases line the interior of the duplex. dBs give them slightly polar character and causes them to hydrogen bond to each other’s polar groups when complementary base pairs are aligned linearly as “wrungs” in the duplex ladder.
A and T bond through 2 H bonds.
G and C bond through 3.
Bonding pairs are always purine/pyrimidine.

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25
Q

Describe how Hershey and Chase showed DNA to be the genetic material.

A

Expt. by Hershey and Chase:
- Viruses were radioactively labelled so either proteins or DNA were detectable
- Cells were infected and lysed.
- Lysate was centrifuged and contents of pellet (the cells) were scanned for radioactive content
Outcome:
- Only the virus with radioactive label in the DNA gave a radioactive cell pellet.
** The DNA was what the virus incorporated into the host and produced the genetically mediated viral infection. Radioactive caspids were in the supernatant (not what conferred the effect in cells).

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26
Q

How was DNA shown to be the genetic material?

A

The results of experiments by Avery, MacLeod, and McCarty: a non-infectious bacterium population was injected with the DNA of an infectious strain. The non virulent strain became infectious.

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27
Q

Why was the base composition (Chargaff’s rules) important in suggesting a model for the structure of DNA?

A

Chargaff & co found that bases occurred in DNA in different proportions and that the proportions of certain bases were related. (%G = %C, %A = %T)
This is supportive of the WC base pairing paradigm, where two certain bases occur jointly and therefore should compose equal proportions of the whole bp population.

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28
Q

What other information was crucial in the structure determination of DNA?

A

Franklin and Wilkins - x-ray crystallography and diffraction = helical with two periodicities (2 groove sizes)

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29
Q

Discuss the structure of double-stranded DNA. (Point out the major and minor grooves, the sugar-phosphate backbones, and the stacked base pairs.)

A

Double helix formed by coiling complementary, anti parallel strands. Bases paired on interior (maintained by h-bonds and hydrophobic forces).
10.5 bp per turn = 36 Angstroms per turn and 3.4 Angstroms between bps.

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30
Q

Define site directed mutagenesis.

A

Method to introduce single residue mutations into an expressed protein based on restriction sites in gene of interest.
Gene can be cleaved at restriction site and an a segment that is identical except a few bp changes can be spliced in based on the same restriction cleavage.

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31
Q

Define oligonucleotide directed mutagenesis.

A
  • A primer (oligonucleotide, 21-40 bp long) is generated complementary to the sequence to be replaced except with the desired bp changes. Complementary primer also created.
  • Gene is denatured with primers and annealed.
  • Primer provides 3’ site for DNAp. DNAp synthesizes new whole plasmid from template and primer.
  • Original primers may be degraded by DpnII if selectively methylated or otherwise marked. Mutant plasmids may be ligated and anneal to form mutant cDNA.
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32
Q

Distinguish oligonucleotide directed mutagenesis from site directed.

A

Oligo uses primers to prompt creation of a whole new plasmid from the cDNA template. Site directed depends on restriction sites around mutation point in order to replace the original, non mutant DNA.

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33
Q

Describe how DNA primers and probes of a specific sequence are synthesized using solid phase synthesis.

A
  • Solid phase or silicon beads are in column.
  • Blocked dNTPs have a DTP, acid labile blocking agent attached to its 5’ end and are fixed by 3’ end to silicon.
  • Acid washed through removes block.
  • Next base to be added: blocked dNTP of that base is washed through column. Excess removed.
  • Acid removed… repeat.
  • Finally, cleavage rxn removes cyanohydrin protective groups and cleaves polypeptide product from silica.
34
Q

Suppose you want to use oligonucleotide directed mutagenesis to make a mutant gene that encodes a protein containing phenylalanine rather than tyrosine. Given the following DNA and protein sequence, write the sequence of the primers you would design to make the mutant protein.
…. Cys Ser Leu Tyr14 Gln Leu Glu…
…. TGT AGT CTC TAT CAA TTA GAA…

A

Original:…. TGT AGT CTC TAT CAA TTA GAA…
New: ……..ACA TCA GAG (phe) GTT AAT CTT ….

Different possible sequences that are an anticodon for Phe can be put in.

35
Q

Describe a procedure used to tag proteins thereby facilitating their purification using affinity chromatography techniques.

A
  • Make a fusion protein gene (restriction enzyme fusion of separate genes) with a specific, well characterized affinity tag on either of the terminals
    Ex: His tag, GST
  • Tag gene fused to protein gene
  • Expressed via expression vector
  • Lysate collected and the tag can be used to isolate, evaluate presence, or see interactions of the tagged proteins based on reversible binding interactions with a precipitate or solid state bound ligand
36
Q

What is needed in order to amplify a segment of DNA using PCR?

A

1) DNA segment from which the sequence will be copied - the template
2) Two oligonucleotide primers that will anneal to ends of the sequence, directed towards the inside of the sequence in the 3’ direction
3) dNTPs that will be used to synthesize the new DNA
4) DNA polymerase - will synthesize the new strands and should be a heat shock resistant strain

37
Q

Discuss the three repeated steps PCR: Heating, Cooling, and Replication.

A

1) Heating - denatures DNA and primers
2) Cooling - anneals primers to DNA
3) Replication - with mild heating, DNAp (Taq) binds to primers and extends from 3’ ends inwards.
Repeating the cycle = exponential amplification of the primer flanked sequence/gene of interest

38
Q

PCR: For each copy of the target sequence that you started with, how many copies do you expect after cycle 10 (assuming that the amplification performs perfectly in each cycle)?

A

After round three, starting with 2 copies of endproduct, you will get 2^(#rounds total-3) copies per each original DNA strand.
Ex: 2(10-3) = 2^7 = 128 copies

39
Q

Explain why the use of DNA polymerase from the bacterium Thermus aquaticus (found in hot springs) made a big improvement for PCR.

A

The bacteria polymerase is used to operating at elevated temperatures and most importantly, will not denature following each round of heating to denature the double stranded DNA. If the DNAp were not heat resistant, the DNAp would need to be replaced following every round.

40
Q

What is an STR? Discuss the use of PCR as a forensic tool.

A

A short tandem repeat sequence: conserved between people and have similarly conserved flanking sequences. However, STRs are different lengths for different people.
- DNA fingerprinting: amplifying a set of known STRs via PCR and then measuring the lengths of the products gives rise to a profile unique to each person

41
Q

Describe how PCR could be used to detect HIV infection at an early stage, including what information about HIV would be needed to begin with.

A
  • qPCR can be used to amplify any HIV DNA that is produced by immune cells due to the infection
  • Degree of infection/amount of virus DNA present can be on the rate at which the anti-HIV gene binding-labile probe is activated in qPCR
  • This diagnostic test requires that you know at least end sequences of the gene coding for part of the virus which you can base your primers on
42
Q

Do problems 5 and 8 on pp. 353 - 354.

A

Primer 1) for complement strand = the same base sequence as the __ bp before the beginning of the gene in the known strand or sequence you are provided with
Primer 2) for the known strand = the complement base pair sequence for __ bp after the end of the gene of interest (complement to the provided strand, oriented so the 3’ primer end is facing towards the inside of the gene)

43
Q

What does RT-PCR sand for and why is it used?

A

Reverse Transcription PCR.
Used to amplify the cDNA that corresponds to an isolated mRNA sample. Good for:
- Determining how protein expression (proportional to mRNA production) varies under certain conditions or (in combination with qPCR) at different time points following an alteration

44
Q

What does qPCR stand for and why is it used? Describe how qPCR works.

A

Quantitative PCR.
Used to measure relative amounts of a DNA present in an original sample from the organism genome or from a cDNA sample.
- Gene of interest will bind complementarily with a fluorescent probe that is binding labile and activated.
- The greater the presence of the replicated gene of interest, the more fluorescence activated.
- The quantification = the rate at which the fluorescence at a specific wavelength reaches a threshold intensity as measured by absorption spectroscopy.

45
Q

What is the meaning of the term “library” to a molecular biologist?

A

Library refers to a collection of cloned DNA from a sample that can be used to study the functions of the genes

46
Q

What is a genomic library?

A

A collection of cloned DNA from the entire organism genome. Largest type of DNA library.

47
Q

What is a cDNA library?

A

A collection of DNA copies of all the mRNA being produced in the organism sample at a specific time or under specific conditions.

48
Q

Describe the synthesis of cDNA.

A

Lysate is mixed with poly-T primers that anneal to poly-A 3’ tail of mRNA that has been post processed in sample cells (no introns). Reverse transcriptase binds and produces DNA from RNA. RNA is degraded by base. DNA is primed from 5’ end by oligo nucleotide known for small segment of the gene. DNA p synthesizes other DNA strand.

49
Q

Describe the synthesis of a genomic library.

A

A collection of cloned DNA from the entire organism genome. Genome is partially digested, (correct size segments) inserted into large segment vectors (BACs, YACs) and cloned. Each cell grows into a colony of clones all producing that same cloned segment of DNA.
Largest type of DNA library.

50
Q

In preparing a genomic library for a given kind of animal or plant, does it matter from which type of cell you prepare the DNA? Why or why not?

A

Genomic library generation depends on restriction sites to cleave genome. If common or well conserved sites are used for the partial digestion, it should not matter. All organisms use DNA, the universal code for genome coding.

51
Q

In preparing a cDNA library, does it matter from which type of cell you prepare the mRNA?

A

Yes, it does matter, because some cells have constitutively transcribed genes (housekeeping genes) which must be negatively regulated, while others only produce mRNA for genes when positive regulation induces transcription. A eukaryote may have a smaller cDNA library at a given time because of this. Even more specifically, different factors in different tissues will influence the mRNA production and can give very different cDNA libraries for different parts of the same organism (inherent in specialized cells)

52
Q

How would a cDNA library prepared from mouse liver cells differ from a cDNA library prepared from mouse thyroid cells?

A

Would be secreting and producing different proteins (different non constitutive mRNAs) suited to different function. Thyroid - secretion of hormones, etc. Liver - secretion of digestive and nutrient processing enzymes.

53
Q

List three types of DNA (i.e. not including genes for specific proteins) which would be present in a complete mouse genomic library which would not be present in a cDNA library made from mouse thyroid cells.

A

(1) Centromere sequences
(2) Telomere sequences
(3) Introns

54
Q

Describe how comparative genomics can be used to determine probable protein function.

A

Comparing gene sequences between organisms and within the genome of one organism to genes of known function and sequence can help suggest a role for the unknown proteins:
- Genes with similar sequence (and therefore, similar motifs) in different species are orthologs; similar sequences in same species are paralogs
- Presence of either suggests that they have similar functions.
Order of genes also can be used to suggest function:
- Similar order of genes in one species to genes in another = synteny.
Synteny suggests similar function of the similarly order genes in the other species.

55
Q

Describe GFP and its variants.

A

Structure: beta barrel with fluorophore in center. Fluorophore made from rearrangement of groups and oxidation of amino acids at center.
GFP fluoresces under blue light. Variants have been made to give fluorescence at all different wavelength ranges of visible spectrum.

56
Q

Discuss how direct and indirect immunofluorescence microscopy can be used to determine the sub-cellular localization of a specific protein.

A

Direct protein tagging:

  • Over expression of a fusion protein that either has a fluorescence tag or an epitope with a commercially available antibody epitope.
  • Non fluorescing or unaltered protein may be localized by secondary antibody labeling specific either to the protein itself or to the epitope. Secondary antibodies have fluorophores fused to them.
57
Q

Describe how fusions with epitope tags can be used to identify interacting proteins.

A

Fusion proteins made with epitope tags can be expressed in the cell and isolated from the lysate with their binding proteins by:

1) precipitating the tagged protein and its bound proteins by binding of the epitope to a precipitate
2) washing the tagged protein complex through a column which has binding sites for the epitope. Can be removed from column afterwards by selective proteolysis of the bound segment.
- can be made even more specific with fewer false positives (proteins wrongly identified as interacting) by using a Tandem Affinity Purification (TAP) tag

58
Q

Describe how the yeast two-hybrid analysis is used to identify interacting proteins.

A

Allows you to identify which proteins (A, B, C, or D) interact with the bait protein of interest, Z

1) Generate a yeast line that produces a bait-Gal4p fused gene that produces the corresponding protein
2) Generate an array of yeast that product “fish” or A, B, C, or D linked to the Gal4p activating domain
3) Cross the strains and plate each resulting hybrid
4) Only hybrids that contain a fish protein that takes the bait will survive, because connection of the binding and activating proteins will result in transcription of the gene that is necessary for the yeast’s survival (selectable marker) or will cause it to present a screenable marker

59
Q

What is a TAP tag and how is it used to more specifically isolate protein complexes to identify interacting protein components?

A

A tandem affinity purification tag is a TAP tag that is fused to the protein of interest (inserted adjacent to gene of interest before cloning) that has two epitopes. The first epitope is used for one round of immunoprecipitation or column chromatography and then cleaved off. Repeating the purification with binding the second epitope only increases purity of the isolated complex.

60
Q

What is a DNA microarray?

A

A device used to screen for thousands of genes at once that might be present in a sample: identifies cDNA that make up the mRNA profile of a sample by hybridizing fluorescently labeled cDNA with complementary sequences of genes of interest that have been fixed to a chip in sectors. Each sector contains many copies of a single stranded complementary DNA.

61
Q

Describe the photolithographic method for fabricating DNA microarrays.

A

Similar to making microchips: blank chip with reactive sector spots has a photoactivated binding agent

1) Genes of interest entered into computer
2) chip washed with solution of activated nucleic acids - either G, A, T, or C - which also have a light sensitive binding agent
3) light is selectively shone on spots where the computer wants the nucleotides to bind
4) chip is washed off
5) Another wash is applied of another nucleic acid wash, light is selectively shone, washed again, and repeat until the sequences in all the spots are assembled

62
Q

Discuss how a DNA microarray (or chip) can be used to study changes in gene expression that accompany development.

A
  • Collect the two samples of mRNA
  • Use reverse transcriptase and base to generate cDNA of the mRNA (a cDNA library) using fluorescent bases in different colors
  • Make a DNA microarray with the desired genes of interest
  • Wash with denatured cDNA so that the single strands of fluorescing cDNA bind to the fixed DNA on the chip
  • Relative amounts of fluorescence on each sector spot reveal the different relative abundance** mRNA expression patterns dependent on the initial conditions (in this case, developmental) of the samples
63
Q

What are micro-RNAs? Explain how RNAi can be used as a gene silencing method.

A

Micro RNAs are RNA transcriptions and can regulate translation by preventing translation or causing the mRNA to be degraded:
1) Transcribed in nucleus as precursor - self complementary and folded
2) transported to cytoplasm, cleaved by endonucleases to activate
3) Activated, ~20 bp mRNAs bind either partially or fully to target mRNA (= delaying translation by binding to ribosome binding sequence or triggering degredation)
RNAi = RNA interference. This is manufacture of small interfering RNAs (siRNAs) that are expressed in the cell as self complementary precursors, processed similarly and act to knockdown mRNA translation of a target gene. RNAi consists of generating this siRNA and applying it to make the KO.

64
Q

Draw the structure of ddATP (you may use A as an abbreviation for the structure of the base).

A

Do dat shit

65
Q

Describe the Sanger method for DNA sequencing.

A

Need: DNA of interest, dideoxynucleosides (ddNTPs), DNAp, DNA helicase, normal dNTPs

  • 4 solutions - each contains only one type of ddNTP and the rest are normal dNTPs
    1) Have DNA split by helicase and primer segment of known sequence (with radioactive or fluorescent label bound)
    2) Synthesize new complementary DNA strands with DNAp, and ddNTPs in four solns.
    3) DNA synthesis will end where the ddNTP of that solution has been inserted
    4) Gel electrophoresis will show the relative lengths of DNA that ends with each certain bp = the sequence of bps in the segment
66
Q

How does incorporation of a dideoxynucleoside affect subsequent polymerization by DNA polymerase?

A

ddNTP does not have the reactive 3’ OH site necessary for further polymerization. Strand synthesis will halt.

67
Q

How many reactions are included in a typical DNA sequencing experiment?

A

4.

1) Annealing of RNA primers
- Primers can have a fluorescent or radioactive label tagged to it**
2) Synthesis of DNA until ddNTP is added
- In original Sanger method, 4 tubes have these rxns
- In modern Sanger method, one tube has fluorescently labeled ddNTPs and all these rxns in one mix.
3) Separation of short strand from template
4) Run combined sample through gel electrophoresis gel or capillary tube to separate synthesized strands according to length

68
Q

What is a radioactive isotope?

A

An unstable isotope of an atom (unstable nucleus) that stabilizes itself by emitting ionizing radiation.

69
Q

Do problem 14 on p. 310.

A

How do the different lengths of the separated synthesized DNA strands correspond to relative location of a given base in the sequence?

70
Q

What is fluorescence?

A

Electromagnetic radiation emitted from a molecule as its electrons return to ground state (after being excited by a wavelength of higher frequency).

71
Q

Compare fluorescence to radioactivity.

A

Fluorescence is light emitted by a stable molecule as excited electrons return to ground state.
Radioactivity is the emission of ionizing radiation from an element with an unstable nucleus.

72
Q

In a more modern and more highly automatable version of the original Sanger method, fluorescent tags can be attached to the dideoxynucleotides to make them detectable by fluorescence measurements. Why is it possible with this method to have only a single reaction mixture?

A

Since the 4 ddNTPs emit unique fluorescent signals (different wavelengths that can be read by the computer) they can be distinguished based on the fluorescence registered, not by simply the presence of radioactivity (as is the case with the original Sanger method, where you must separate the solutions into four lanes).

73
Q

Modern Sanger method: After separating the fragments, how do you know which fragment stopped at which residue?

A

Based on the length of the fragment and the fluorescence wavelength of the fragment = which ddNTP the fragment ended with.

74
Q

Modern Sanger method: Why does this method allow more sequence information to be derived from a single sequencing reaction?

A

You can learn all the sequencing info from the reaction taking place in one tube?

75
Q

Describe next-generation pyrosequencing.

A

Tracks DNA sequence by addition of a certain dNTP to a segment = creation of pyrophosphate (by DNA polymerase) = creation of ATP (by sulfurylase) = flash of light (reaction of ATP with luciferin by luciferase).
One dNTP type is washed over solid state, single stranded DNA template, so each flash of light (with particular intensity and location on the plate) corresponds to a certain base (in a certain number) being complementary to a sequence (at a certain location on the plate).
Excess dNTPs after each wash are quickly degraded by apyase.

76
Q

Describe next-generation reversible terminator sequencing.

A
  • Allows you to tell what dNTP has been added to a given segment based on the fluorescence emission
  • Fluorescently labeled, reversibly blocked dNTPs are added ONE AT A TIME (due to blocking) to 3’ end of primer that has annealed to a known oligonucleotide that has been fused to the template DNA
  • Following one addition, the DNA there will fluoresce according to the unique fluorscence (red, green, blue, orange for G, A, T, and C, for example) that has been added
  • Computer flashes laser to read fluorescent signal at each segment location
  • Blocking and fluorescence removed from dNTP most recently added
  • New fluorescent, blocking dNTPs washed in
  • Repeat
77
Q

How many genes in the human genome?

A

~ 25,000 coding for proteins

about twice that of a fly, and fewer than a rice plant

78
Q

What fraction of the human genome consists of genes?

A

~ 30% (exons and introns)

79
Q

What fraction of the genome encodes protein?

A

~1.5% (exons alone)

80
Q

What are transposons? What fraction of the human genome do they represent?

A

~45%.
These are sequences that act as molecular parasites and essentially copy themselves and move around in the genome of basically every organism.
- The products of their transcription and translation can catalyze their own movement

81
Q

Define SSR and SNP.

A

SSR - short sequence repeat. These sequences are small (10 bp about) repeated sequences that are implied in cellular metabolism and centromere function in humans.
SNP - Single Nucleotide Polymorphisms. Occur about every 1/1000 bp for humans. Single bp changes between humans. Give rise to familiar variations between people, like hair color, height, allergies, and to some extent, behavior.

82
Q

How do the short sequences generated using reversible termination sequencing assemble into contigs?

A

DNA segments prepared from sample as chunks with overlapping regions. As segments are sequenced, computer algorithmically uses the overlaps to assemble the segments into long, contiguous strands called contigs.

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