BIOCHEM - Lecture 4 - Macromolecule Characterization Flashcards

1
Q

DNA gels:

DNA fragments can be separated easily based on _____ (bc ___ is constant) using an _____ gel electrophoresis

A

mass (bc m/z is constant) using an agarose gel electrophoresis

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2
Q

DNA gels:

DNA bands are visualized by staining with ___ ___

A

ethidium bromide

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3
Q

doxorubin is an ___-____ agent

it ____ into ____, disrupting its ____ and ____

A

anti-cancer agent

intercalates into DNA, disrupting its structure and function

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4
Q

mechanism of doxorubicin-DNA complex:

  1. doxorubicin forms a ____ bond with ____ on one strand of DNA through a _____-mediated reaction
  2. ____ bonds ____ its interaction with the ____ strand
A
  1. covalent bond with guanine on one strand of DNA through a formaldehyde-mediated reaction
  2. hydrogen bonds stabilize its interaction with the opposing strand
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5
Q

mechanism of doxorubicin-DNA complex:

doxorubicin ____ into DNA and pushes apart the flanking ___ ___ with the ___ ____ sitting in the ____ groove

A

intercalates into DNA and pushes apart the flanking base pairs with the sugar moiety sitting in the minor groove

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6
Q

mechanism of doxorubicin-DNA complex:

cancer cells often exhibit higher rates of ____

they ____ more rapidly than normal cells, and DOX-induced damage is more lethal to ___ ____ cells

A

proliferation

divide more rapidly than normal cells, and DOX-induced damage is more lethal to rapidly dividing cells

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7
Q

mechanism of doxorubicin-DNA complex:

cancer cells have compromised ___ ___ ____, making them more susceptible to DNA ____ caused by DOX

A

DNA repair mechanisms, making them more susceptible to DNA damage caused by DOX

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8
Q

DNA restriction endonucleases:

endonucleases are enzymes that recognize specific ____ ____ through ___ ___-____, and ___ the DNA at those sites

A

DNA sequences through side-chain H-bonds, and cleave the DNA at those sites

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9
Q

DNA restriction endonucleases:

restriction enzymes cut at ____ ____, which ensures cuts from ____ ____

A

palindromic sequences, which ensures cuts from both directions

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10
Q

DNA restriction endonucleases:

some enzymes cleave the DNA to produce ____ ____ (___ cuts), and others generate ___ ___ (___ cuts)

A

sticky ends (staggered cuts), and others generate blunt ends (straight cuts)

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11
Q

DNA restriction endonucleases:

EcoRI alpha-helix inserts into DNA ___ ____

A

major groove

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12
Q

DNA restriction endonucleases:

endonucleases cut their specific sequence through a ____ ____ cleavage mechanism

restriction enzymes ____ a ____ ____ transition state

this facilitates ____ by a ___ molecule, breaking the DNA ____ ___

____ ___ and ____ ___ in the enzyme’s ____ site assist in catalysis

A

phosphodiester bond cleavage mechanism

stabilize a pentavalent phosphate transition state

cleavage by a water molecule, breaking the DNA phosphate backbone

magnesium ions and aspartate residues in the enzyme’s active site assist in catalysis

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13
Q

cloning DNA in bacteria:

cloning involves the insertion of ___ ___ into ____, which are then introduced into ___ ____ (like E. coli)

A

DNA fragments into plasmids, which are then introduced into bacterial cells (like E. coli)

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14
Q

cloning DNA in bacteria:

____ and ___ ___ can be used to ___ and ____ DNA ____ into _____

A

endonucleases and DNA ligase can be used to cut and paste DNA sequences into plasmids

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15
Q

cloning DNA in bacteria:

plasmids can be ____ in ____ and then ____ to obtain milligrams of ___ per liter of ____

A

amplified in E. coli and then purified to obtain milligrams of DNA per liter of culture

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16
Q

cloning DNA in bacteria:

  1. chromosomal DNA contains the ___ of interest

___ different ___ ____ are used

one cuts at a palindromic sequence, producing ____ ends

the other cuts at a different sequence, producing ___ ends

the plasmid ___ ___ is also cut with the same enzymes to allow for the ____ of the DNA fragment

A
  1. gene of interest

2 different restriction enzymes are used

sticky ends

blunt ends

cloning vector is also cut with the same enzymes to allow for the insertion of the DNA fragment

17
Q

cloning DNA in bacteria:

  1. the cut DNA fragment is inserted into the ____ ____, which has:

a ___ ___ (so it can ___ in bacteria)

an ___ ___ ___ (used for selection)

DNA ____ joins the DNA fragment and the plasmid tg

A

plasmid vector, which has:

replication origin (so it can replicate in bacteria)

ampicillin resistance gene (used for selection)

ligase joins the DNA fragment and the plasmid tg

18
Q

cloning DNA in bacteria:

the ____ plasmid is introduced into E. coli cells via ____

bacteria that take up the plasmid will have the ___ ___ ___

A

recombinant plasmid is introduced into E. coli cells via transformation

bacteria that take up the plasmid will have the ampicillin resistance gene

19
Q

cloning DNA in bacteria:

the bacteria are grown on an ____-containing plate

only bacteria that successfully incorporated the plasmid will ____, forming colonies

A

ampicillin-containing plate

survive, forming colonies

20
Q

cloning DNA in bacteria:

plasmids are ____ from bacterial cultures

the presence of the inserted DNA is confirmed by ___ ____

A

gel electrophoresis

21
Q

DNA synthesis review:

____ ____ are essential for DNA sequencing and ____

A

synthetic oligonucleotides are essential for DNA sequencing and mutagenesis

22
Q

DNA synthesis review:

  1. the first nucleoside is attached to a ___ ___ (___) at the ___ ____ (___) position

the ___ ___ is protected by a _____ group to prevent unwanted reactions

A
  1. solid support (resin) at the 3’ hydroxyl (OH) position

5’ hydroxyl is protected by a DMT group to prevent unwanted reactions

23
Q

DNA synthesis review:

  1. the ___ group is chemically removed, exposing the ___ ____, so that the next ___ can be ____
A

DMT group is chemically removed, exposing the 5’ hydroxyl, so that the next nucleotide can be added

24
Q

DNA synthesis review:

  1. the next nucleotide is ____ at its ___ ___ via a _____ group

a chemical reaction links the incoming nucleotide’s ___ ___ to the previous nucleotide’s ___ ___, forming a ____-like bond

____ is released as a byproduct

A

activated at its 3’ phosphate via a phosphoramidite group

5’ hydroxyl to the previous nucleotide’s 3’ position, forming a phosphodiester-like bond

diisopropylamine is released as a byproduct

25
DNA synthesis review: 4. the new phosphite linkage is ____ to a stable ___ ____, ensuring a strong backbone
oxidized to a stable phosphate triester, ensuring a strong backbone
26
DNA synthesis review: steps 2-4 are repeated until the ___ ___ ___ is synthesized / all ____ are added
full DNA sequence is synthesized / all residues are added
27
DNA synthesis review: 5. ___ groups on the DNA bases (___ groups for nucleobase nitrogens) are removed 6. ____ groups on phosphates are removed 7. ____ ___ is cleaved from the ____ ____, completing synthesis
protecting groups on the DNA bases (benzoyl groups for nucleobase nitrogens) are removed cyanoethyl groups on phosphates are removed DNA chain is cleaved from the silica support, completing synthesis
28
DNA synthesis review: DNA primers are typically ___-___ nucleotides long the ___-___ glycosidic bond in ___ (__ and ___) is ____-sensitive, so (too) strong acids can cause ____, leading to "___" sites (loss of ____)
20-40 nucleotides long C-N glycosidic bond in purines (A and G) is acid-sensitive, so (too) strong acids can cause depurination, leading to "abasic" sites (loss of bases)
29
DNA synthesis review: a not-shown "step 4b" involves adding __-____ via _____
5'-phosphate via phosphoramidite
30
DNA sequencing: dideoxynucleotides (ddNTPS): primers are elongated by ___ ___ with a nucleotide mixture with ___ ____ ____ (____)
DNA polymerase with a nucleotide mixture with 4 fluorescent dideoxynucleotides (ddNTPs)
31
DNA sequencing: dideoxynucleotides (ddNTPS): each synthesis terminates with a _____ (lacking ___-___ ___) the mixture is then separated by ___ ____ and analyzed by ___-____ ____
ddNTP (lacking 3'-OH group) gel electrophoresis and analyzed by laser-excited fluorescence
32
____ DNA sequencing is still the "gold standard"
sanger DNA sequencing is still the "gold standard"
33
sanger DNA sequencing: 1. a short ____ binds to the single-stranded DNA ____ to help DNA ____ start replication DNA polymerase extends the strand using: - regular _____ (____) - fluorescently labeled ____ (____) - these lack a ___-____ group, so when they're incorporated, DNA synthesis ____ at that position
primer binds to the single-stranded DNA template to help DNA polymerase start replication - nucleotides (dNTPS) - dideoxynucleotides (ddNTPs) - these lack a 3'-OH group, so when they're incorporated, DNA synthesis stops at that position
34
sanger DNA sequencing: 2. since ddNTPs randomly incorporate at different positions, the result is a mixture of DNA fragments that all ____ at ___ ____ each fragment is fluorescently labeled according to the ____ that caused _____
end at different nucleotides ddNTP that caused termination
35
sanger DNA sequencing: 3. the DNA fragments are ____ (separated from ___ ___; made ___-___) and loaded into a capillary gel for ____ smaller fragments migrate ____, while longer fragments move ___ a ___ ___ detects the ___ as each fragment passes through the detector
denatured (separated from template strand; made single-stranded) and loaded into a capillary gel for electrophoresis faster, while longer fragments move slower laser beam detects the fluorescence as each fragment passes through the detector
36
sanger DNA sequencing: 4. computer records the ___ of each detected fragment and aligns them based on ____ each peak on the computer represents a specific ____ the order of peaks reveals the ____ of the ___ ___ ____
color of each detected fragment and aligns them based on size nucleotide sequence of the original DNA strand
37