BIOCHEM - Lecture 4 - Macromolecule Characterization Flashcards
DNA gels:
DNA fragments can be separated easily based on _____ (bc ___ is constant) using an _____ gel electrophoresis
mass (bc m/z is constant) using an agarose gel electrophoresis
DNA gels:
DNA bands are visualized by staining with ___ ___
ethidium bromide
doxorubin is an ___-____ agent
it ____ into ____, disrupting its ____ and ____
anti-cancer agent
intercalates into DNA, disrupting its structure and function
mechanism of doxorubicin-DNA complex:
- doxorubicin forms a ____ bond with ____ on one strand of DNA through a _____-mediated reaction
- ____ bonds ____ its interaction with the ____ strand
- covalent bond with guanine on one strand of DNA through a formaldehyde-mediated reaction
- hydrogen bonds stabilize its interaction with the opposing strand
mechanism of doxorubicin-DNA complex:
doxorubicin ____ into DNA and pushes apart the flanking ___ ___ with the ___ ____ sitting in the ____ groove
intercalates into DNA and pushes apart the flanking base pairs with the sugar moiety sitting in the minor groove
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
proliferation
divide more rapidly than normal cells, and DOX-induced damage is more lethal to rapidly dividing cells
mechanism of doxorubicin-DNA complex:
cancer cells have compromised ___ ___ ____, making them more susceptible to DNA ____ caused by DOX
DNA repair mechanisms, making them more susceptible to DNA damage caused by DOX
DNA restriction endonucleases:
endonucleases are enzymes that recognize specific ____ ____ through ___ ___-____, and ___ the DNA at those sites
DNA sequences through side-chain H-bonds, and cleave the DNA at those sites
DNA restriction endonucleases:
restriction enzymes cut at ____ ____, which ensures cuts from ____ ____
palindromic sequences, which ensures cuts from both directions
DNA restriction endonucleases:
some enzymes cleave the DNA to produce ____ ____ (___ cuts), and others generate ___ ___ (___ cuts)
sticky ends (staggered cuts), and others generate blunt ends (straight cuts)
DNA restriction endonucleases:
EcoRI alpha-helix inserts into DNA ___ ____
major groove
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
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
cloning DNA in bacteria:
cloning involves the insertion of ___ ___ into ____, which are then introduced into ___ ____ (like E. coli)
DNA fragments into plasmids, which are then introduced into bacterial cells (like E. coli)
cloning DNA in bacteria:
____ and ___ ___ can be used to ___ and ____ DNA ____ into _____
endonucleases and DNA ligase can be used to cut and paste DNA sequences into plasmids
cloning DNA in bacteria:
plasmids can be ____ in ____ and then ____ to obtain milligrams of ___ per liter of ____
amplified in E. coli and then purified to obtain milligrams of DNA per liter of culture
cloning DNA in bacteria:
- 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
- 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
cloning DNA in bacteria:
- 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
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
cloning DNA in bacteria:
the ____ plasmid is introduced into E. coli cells via ____
bacteria that take up the plasmid will have the ___ ___ ___
recombinant plasmid is introduced into E. coli cells via transformation
bacteria that take up the plasmid will have the ampicillin resistance gene
cloning DNA in bacteria:
the bacteria are grown on an ____-containing plate
only bacteria that successfully incorporated the plasmid will ____, forming colonies
ampicillin-containing plate
survive, forming colonies
cloning DNA in bacteria:
plasmids are ____ from bacterial cultures
the presence of the inserted DNA is confirmed by ___ ____
gel electrophoresis
DNA synthesis review:
____ ____ are essential for DNA sequencing and ____
synthetic oligonucleotides are essential for DNA sequencing and mutagenesis
DNA synthesis review:
- the first nucleoside is attached to a ___ ___ (___) at the ___ ____ (___) position
the ___ ___ is protected by a _____ group to prevent unwanted reactions
- solid support (resin) at the 3’ hydroxyl (OH) position
5’ hydroxyl is protected by a DMT group to prevent unwanted reactions
DNA synthesis review:
- the ___ group is chemically removed, exposing the ___ ____, so that the next ___ can be ____
DMT group is chemically removed, exposing the 5’ hydroxyl, so that the next nucleotide can be added
DNA synthesis review:
- 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
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
DNA synthesis review:
- the new phosphite linkage is ____ to a stable ___ ____, ensuring a strong backbone
oxidized to a stable phosphate triester, ensuring a strong backbone
DNA synthesis review:
steps 2-4 are repeated until the ___ ___ ___ is synthesized / all ____ are added
full DNA sequence is synthesized / all residues are added
DNA synthesis review:
- ___ groups on the DNA bases (___ groups for nucleobase nitrogens) are removed
- ____ groups on phosphates are removed
- ____ ___ 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
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)
DNA synthesis review:
a not-shown “step 4b” involves adding __-____ via _____
5’-phosphate via phosphoramidite
DNA sequencing: dideoxynucleotides (ddNTPS):
primers are elongated by ___ ___ with a nucleotide mixture with ___ ____ ____ (____)
DNA polymerase with a nucleotide mixture with 4 fluorescent dideoxynucleotides (ddNTPs)
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
____ DNA sequencing is still the “gold standard”
sanger DNA sequencing is still the “gold standard”
sanger DNA sequencing:
- 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
sanger DNA sequencing:
- 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
sanger DNA sequencing:
- 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
sanger DNA sequencing:
- 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
