Lecture 4 - Macromolecule Characterization (pt. 1)

Question 1

DNA gels:

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

Answer 1

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

Question 2

DNA gels:

DNA bands are visualized by staining with ___ ___

Answer 2

ethidium bromide

Question 3

doxorubin is an ___-____ agent

it ____ into ____, disrupting its ____ and ____

Answer 3

anti-cancer agent

intercalates into DNA, disrupting its structure and function

Question 4

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

Answer 4

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

Question 5

mechanism of doxorubicin-DNA complex:

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

Answer 5

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

Question 6

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

Answer 6

proliferation

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

Question 7

mechanism of doxorubicin-DNA complex:

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

Answer 7

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

Question 8

DNA restriction endonucleases:

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

Answer 8

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

Question 9

DNA restriction endonucleases:

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

Answer 9

palindromic sequences, which ensures cuts from both directions

Question 10

DNA restriction endonucleases:

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

Answer 10

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

Question 11

DNA restriction endonucleases:

EcoRI alpha-helix inserts into DNA ___ ____

Answer 11

major groove

Question 12

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

Answer 12

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

Question 13

cloning DNA in bacteria:

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

Answer 13

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

Question 14

cloning DNA in bacteria:

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

Answer 14

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

Question 15

cloning DNA in bacteria:

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

Answer 15

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

Question 16

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

Answer 16

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

Question 17

cloning DNA in bacteria:

2. 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

Answer 17

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

Question 18

cloning DNA in bacteria:

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

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

Answer 18

recombinant plasmid is introduced into E. coli cells via transformation

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

Question 19

cloning DNA in bacteria:

the bacteria are grown on an ____-containing plate

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

Answer 19

ampicillin-containing plate

survive, forming colonies

Question 20

cloning DNA in bacteria:

plasmids are ____ from bacterial cultures

the presence of the inserted DNA is confirmed by ___ ____

Answer 20

gel electrophoresis

Question 21

DNA synthesis review:

____ ____ are essential for DNA sequencing and ____

Answer 21

synthetic oligonucleotides are essential for DNA sequencing and mutagenesis

Question 22

DNA synthesis review:

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

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

Answer 22

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

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

Question 23

DNA synthesis review:

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

Answer 23

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

Question 24

DNA synthesis review:

3. 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

Answer 24

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

Question 25

DNA synthesis review: 4. the new phosphite linkage is ____ to a stable ___ ____, ensuring a strong backbone

Answer 25

oxidized to a stable phosphate triester, ensuring a strong backbone

Question 26

DNA synthesis review: steps 2-4 are repeated until the ___ ___ ___ is synthesized / all ____ are added

Answer 26

full DNA sequence is synthesized / all residues are added

Question 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

Answer 27

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

Question 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 ____)

Answer 28

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)

Question 29

DNA synthesis review: a not-shown "step 4b" involves adding __-____ via _____

Answer 29

5'-phosphate via phosphoramidite

Question 30

DNA sequencing: dideoxynucleotides (ddNTPS): primers are elongated by ___ ___ with a nucleotide mixture with ___ ____ ____ (____)

Answer 30

DNA polymerase with a nucleotide mixture with 4 fluorescent dideoxynucleotides (ddNTPs)

Question 31

DNA sequencing: dideoxynucleotides (ddNTPS): each synthesis terminates with a _____ (lacking ___-___ ___) the mixture is then separated by ___ ____ and analyzed by ___-____ ____

Answer 31

ddNTP (lacking 3'-OH group) gel electrophoresis and analyzed by laser-excited fluorescence

Question 32

____ DNA sequencing is still the "gold standard"

Answer 32

sanger DNA sequencing is still the "gold standard"

Question 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

Answer 33

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

Question 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 _____

Answer 34

end at different nucleotides ddNTP that caused termination

Question 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

Answer 35

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

Question 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 ___ ___ ____

Answer 36

color of each detected fragment and aligns them based on size nucleotide sequence of the original DNA strand

Question 37

next-gen sequencing: massive ____ of sequencing slightly ___ accurate than sanger, so small sequences of interest can then be ____ using sanger sequencing methods

Answer 37

parallelization of sequencing less accurate than sanger, so small sequences of interest can then be validated using sanger sequencing methods

Question 38

next-gen sequencing: 1. ____ library 2. clonal ____ 3. ____ library 4. ____ ____

Answer 38

1. construct library 2. clonal amplification 3. sequence library 4. analyze data

Question 39

genomic sequences: the ____ of ____ does not scale with genome ___ ( total amount of DNA in its ___) and organism ____

Answer 39

number of genes does not scale with genome size (total amount of DNA in its cells) and organism complexity

Question 40

genomic sequences: the vast majority of human DNA does not code for ___ ____ or ____

Answer 40

functional RNA or proteins

Question 41

polymerase chain reaction (PCR): 1. ____ strands by ____, ____, and ___ ____

Answer 41

separate strands by heating, cool, and anneal primers

Question 42

polymerase chain reaction (PCR): 2. add ____, and extend by ___ ____ gives ____-____ strands

Answer 42

dNTPs, and extend by DNA polymerase variable-length strands

Question 43

polymerase chain reaction (PCR): 3. ____ strands by ____, ____, and ___ ___ ____

Answer 43

separate strands by heating, cool, and anneal more primers

Question 44

polymerase chain reaction (PCR): 4. add ___ and extend ____ by ___ ____ gives ___-____ strands

Answer 44

dNTPs and extend primers by DNA polymerase unit-length strands

Question 45

polymerase chain reaction (PCR): DNA can also be amplified in ___ using purified ____ in PCR ___ polymerase can extend ____ by several ____ __ kb/min taq polymerase has low ____, meaning it can ___ ___ before completing synthesis

Answer 45

vitro using purified polymerase in PCR taq polymerase can extend primer by several kilobases 1 kb/min processivity meaning it can fall off before completing synthesis

Question 46

polymerase chain reaction (PCR): the ____ of polymerases like ___ is essential to a simple PCR process with heating up to ___ degrees celsius

Answer 46

thermostability of polymerases like Taq is essential to a simple PCR process with heating up to 95 degrees celsius

Question 47

polymerase chain reaction (PCR): after ____ cycles, 10^__-fold amplification

Answer 47

25 cycles, 10^6-fold amplification

Question 48

mutating DNA: PCR can be used to ____ sequences with a few ____ by using ___ that contain the ____ the resulting ____ is then ____ in E. coli and then the ___ is ____ and ____

Answer 48

amplify sequences with a few mutations by using primers that contain the mutations plasmid is then amplified in E. coli and then the plasmid is purified and sequenced

Question 49

mutating DNA: ____ DNA (original ___) is degraded by ____, leaving only the ___ sequence as the next ____

Answer 49

methylated DNA (original plasmid) is degraded by Dpnl, leaving only the mutated sequence as the next template

Question 50

mutating DNA: 1. a synthetic oligonucleotide ___ is designed to contain the desired ____ (___ changes) the primer ____/___ to the target DNA, which is a ___ containing the ____ to be altered

Answer 50

primer is designed to contain the desired mutations (base changes) binds/anneals to the target DNA, which is a plasmid containing the gene to be altered

Question 51

mutating DNA: 2. DNA ____ extends the ___ ____, incorporating the ____ into a newly synthesized strand of DNA this generates a ____ ___ within the ____

Answer 51

polymerase extends the mismatched primer, incorporating the mutations into a newly synthesized strand of DNA mutated gene within the plasmid

Question 52

mutating DNA: 3. the original plasmid is ____ (naturally occurring in bacteria) the enzyme ____ specifically degrades ___ ____, meaning that only the newly synthesized (___) DNA remains

Answer 52

methylated (naturally occurring in bacteria) Dpnl specifically degrades methylated DNA, meaning that only the newly synthesized (mutated) DNA remains

Question 53

mutating DNA: 4. the ___ plasmid is introduced into E. coli cells via ____ the bacteria ____ the plasmid, allowing for ___ and ____

Answer 53

mutated plasmid is introduced into E. coli cells via transformation replicate the plasmid, allowing for selection and amplification

Question 54

mutating DNA: 5. colonies containing the ____ plasmid are selected and grown plasmid DNA is extracted and ___ to confirm the presence of the ___ ____

Answer 54

mutated plasmid are selected and grown sequenced to confirm the presence of the intended mutation

Question 55

protein sequences from DNA sequences: protein sequences can be easily determined if the ___ ___ for a protein is known via the ___ ___

Answer 55

DNA sequence for a protein is known via the genetic code

Question 56

protein sequences from DNA sequences: ___ ___ projects and software for ___ the ___ ____ that are converted to ___ have made obtaining ___ ___ trivial

Answer 56

genome sequencing projects and software for identifying the DNA sequences that are converted to protein have made obtaining primary sequences trivial

Question 57

protein sequences from DNA sequences: obtaining primary sequence data from the DNA requires knowing which ____ ___ corresponds to the ___ of ____ this is not always the case as genes have complex ____, ___ elements, and ___ ____

Answer 57

DNA region corresponds to the protein of interest structures, regulatory elements, and alternative splicing