Week 3 (PCR and RNA Review) Flashcards
1
Q
PCR
A
Polymerase Chain Reaction
2
Q
what steps are involved to prepare DNA for replication?
A
- DNA unwinded from histones
- separation of the DNA strands (unzipped)
- primase/RNA primer added
- DNA polymerase must be able to come in and make the new DNA strand
3
Q
why is an RNA primer so important?
A
it makes a FREE 3’ hydroxyl available for DNA polymerase to bind
4
Q
what adds a free 3’ hydroxyl group so that DNA polymerase can bind?
A
primer
5
Q
In which direction is DNA synthesized?
A
5’ to 3’
6
Q
on a forward facing DNA strand (5’—- 3’), where would the primer need to be placed?
A
toward the 3’ end because DNA is read 3’ to 5’ so that it can be synthesized 5’ to 3’
7
Q
on a reverse facing DNA strand (3’—- 5’), where would the primer need to be placed?
A
toward the 3’ end because DNA is read 3’ to 5’ so that it can be synthesized 5’ to 3’
8
Q
when will DNA polymerase stop reading the DNA strand?
A
when there is no longer a template to read from
9
Q
requirements for replicating DNA
A
- DNA template
- DNA polymerase
- nucleotides (dNTP)
- primers
10
Q
what is the equation for number of copies of target?
A
- N*2^C
- N = number of starting copies
- C = number of cycles
11
Q
N*2^C
A
number of copies of target strand
12
Q
N*2^C - what does N mean?
A
number of starting copies
13
Q
N*2^C - what does C mean?
A
number of cycles
14
Q
ex: N*2^C, you have 1 starting copy and 2 cycles
A
4
15
Q
ex: N*2^C, you have 1 starting copy and 25 cycles
A
33.5M
16
Q
Why do you lose some DNA every time you replicate?
A
the primer attaches to the DNA template and will start copying at your target DNA, it is only trying to amplify your target DNA
17
Q
What are the 3 steps in PCR?
A
- denaturation
- annealing
- extension
18
Q
what occurs during the first step of PCR?
A
- denaturation
- 94 degrees Celsius
- melt the two strands apart
19
Q
what temperature does it need to be for denaturation?
A
94 degrees C
20
Q
why do we heat up the DNA to separate the 2 strands?
A
weak hydrogen bonds hold them together and so they can be separated by heat
21
Q
what can be used to separate the DNA from the histones?
A
phenol
22
Q
what occurs during the second step of PCR?
A
- annealing of primers
- 54 degrees Celsius
- warm enough to keep DNA from coming back together but cool enough to have primers bind to each single strand of DNA
23
Q
what temperature does it need to be for annealing?
A
54 degrees Celsius
24
Q
why does it need to be 55 degrees Celsius during the annealing process?
A
warm enough to keep DNA from coming back together but cool enough to have primers bind to each single strand of DNA
25
why does the temperature need to cool down rapidly between denaturation and annealing?
if it is done slowly, you will have nonspecific priming
26
what occurs during the third step of PCR?
- extension
- 72 degrees Celsius
- optimal temperature for DNA polymerase
- extend in the 5' to 3' direction
27
what temperature does it need to be for extension?
72 degrees celsius
28
why does it need to be 72 degrees celsius during extension?
optimal temperature for DNA polymerase
29
what is an example of a DNA polymerase used in PCR? Where does it originate from?
- TAQ
- it comes from a bacteria that lives in a extremely hot water so it has a DNA polymerase that can replicate in hot temperatures
30
what are the 3 things that are key to PCR success?
1. primer specificity
2. annealing temp
3. Mg2+ concentration
31
what affects the primer specificity?
- determined by the length of the primer
- estimated as 4^k (k is the number of bases in the primer)
32
what is the k in 4^k?
the number of bases in the primer
33
for primer specificity, what does it mean if k=4?
there are 4 bases in the primer, so there is 1 in 256 bases
34
how does annealing temperature affect specificity (low vs high temp)?
- low temp = less specific
- high temp = more specific
35
how does Mg2++ concentration affect specificity (low vs high concentration)?
- low Mg = higher specificity
- high MG = lower specificity
36
what is the minimum primer length to assume that it only binds to one location in the genome?
between 15 and 17
37
when you do a PCR and get several lines light up what is this called? Why is it bad?
nonspecificity, you did not appropriately set up the "recipe" right, and it amplified more than the gene that you wanted
38
What would a really good PCR look like?
distinct bands
39
What do you do if you run PCR for the first time and you get non-specificity? What options do you have?
- increase the annealing temperature (so the primers will anneal to the place that it is targeting)
- decrease magnesium
40
when would you increase temperature in the PCR to be more specific?
during annealing so the primers will anneal to the place that it is targeting
41
how do you solve what the optimal PCR is? You have 2 variables.
Hold one variable constant and change the other
42
when finding the optimal PCR, how can you speed up the process?
using multiple thermal cyclers
43
do you have to optimize for every primer?
yes
44
what is a Poly A tail?
a chain of adenine nucleotides added to the end of a messenger RNA (mRNA) molecule. The poly(A) tail helps stabilize the mRNA and allows it to be exported from the nucleus to the cytoplasm
45
what primer can you add, knowing you have an RNA with a Poly A tail?
an oligo dT primer
46
What is needed for a polymerase to bind to the molecule?
free 3' hydroxyl
47
what molecule do we need to read RNA and make a new strand of DNA?
reverse transcriptase
48
RT-PCR
Reverse Transcriptase Polymerase Chain Reaction
49
what is the purpose of RT-PRC?
synthesizing a new strand of DNA from mRNA
50
some limitations of PCR
- size
- base complexity
- secondary structure
51
why are you limited to the size of the DNA segment you can copy in PCR?
polymerases have a process severity rate (they add nucleotides at a certain rate), if you have to heat up and cool down the PCR over and over again polymerases will lose their activity
52
in general using specialized polymerases one can get about _____ kb
30
53
why does base complexity limit PCR?
repetitive sequences are a problem for primer specificity
54
how can a high content of Gs and Cs affect PCR?
they have 3 hydrogen bonds between them so you will need more heat to break apart the DNA
55
how can a high content of As and Ts affect PCR?
you need less heat to separate the strands because there is only 2 hydrogen bonds between them, however at lower temperatures primers are going to anneal to non specific locations
56
examples of a secondary structure and its shape
| a secondary structure of RNA
tRNAs, clover leaf
57
is it easier or harder for a DNA polymerase to get through secondary structures?
it will be more difficult (think about a polymerase trying to go through a hairpin/cloverleaf)
58
what is the difference between dTTP and ddTTP?
dTTP (deoxythymidine triphosphate) has a free 3' hydroxyl and ddTTP (dideoxythymidine triphosphate) does not have a hydroxyl, only a hydrogen. This makes ddTTP a terminator because a polymerase cannot add another nucleotide after it is added, it has to stop.
59
sanger sequencing vs PCR?
in PCR there are all 4 nucleotides in every reaction, in sanger sequencing there are 4 tubes (one for each nucleotide), run 4 PCR
60
in sanger sequencing, what does each of the 4 tubes contain?
1. DNA fragment to be sequenced
2. primer
3. dNTP (bases A, G, T, C)
4. polymerase
5. ddNTP
61
ddNTP
dideoxynucleotide triphosphate
62
what is a terminator?
- ddNTP
- when DNA polymerases use the ddNTP the process stops
63
there is a _____ ratio of ddNTP to dNTP so you don't stop at only the first point they appear
low
64
how did they make reading sanger sequences more efficient?
add fluorescent dye to each one of the dideoxynucleotides so that each nucleotide fluorescents at different wavelengths, this allows a machine to read it (normal nucleotides are unlabeled)
65
what did the Human Genome Project use to sequence the human genome?
dideoxy-sequencing method
66
dideoxy-sequencing method can be _____________, meaning humans spend less time analyzing the results
automated
67
central dogma
DNA-->RNA-->Protein
68
where does transcription occur in the cell?
the nucleus
69
where does translation occur in the cell?
the cytoplasm
70
key components needed for transcription
1. a DNA template
2. the raw materials (ribonucleotide triphosphates - A,C,G,U) needed to build a new RNA molecule
3. the transcription apparatus, consisting of the proteins necessary for catalyzing the synthesis of RNA
71
what has to happen first to DNA in order for a gene to be transcribed?
nucleosome repositioning --> the DNA must be made accessible, meaning it must be separated from histones
72
why is the nontemplate strand of DNA not usually transcribed?
it would make a different protein, the strand is complementary
73
RNA is synthesized in the ____' to ___' direction
3' to 5'
74
during transcription, DNA is read in the ___' to ___' direction
3' to 5'
75
what are two differences between DNA replication and transcription?
1. incoming nucleotides have and OH at 2'
2. RNA uses Uracil
76
Which is the 5' and 3' end of DNA?
this is because the polymerase started on the 3' end, so as it synthesized towards the 5' end there is added length (added nucleotides)
77
Why is it that simple organisms have approximately the same number of protein coding genes as complex organisms?
it has to do with regulation of those genes. you can produce many proteins from the same template.
78
the number of genes ________ strongly correlated with organismal complexity
IS NOT
79
T2T
telomere to telomere
80
when was the first genome read telomere to telomere, when was the human genome project really finished?
2022
81
a lot of what is transcribed and not translated can have a lot of ____________ roles
regulatory
82
genes can be turned on and off depending on:
- time in development
- nutrients
- stress
83
what do regulatory regions of a gene determine?
if transcription occurs: the what, when, where, how much
84
the what, when, where, how much of translation
- what: gene needs to be turned on
- when: what point in does it need to be turned on
- where: which tissue, which cell type
- how much: amount (a little vs a lot)
85
what are control structures in transcription?
- regulatory promoters
- core promoters
- transcription factors
- enhancers
86
regulatory promoters
- upstream of the core promoter
- transcription factors can bind here
- affect the RATE of transcription
87
regulatory promoter affect the _____ of transcription
RATE
88
enhancers
distal locations can also enhance transcription
89
core promoter
where polymerase binds
90
what is upstream on the gene?
before the promoter
91
what is downstream on the gene?
after the promoter
92
_____ is the fundamental unit of heredity
gene
93
if the promoter/enhancers/regulatory proteins "say so", a protein-coding gene is ____________, and the result is a "___________"
transcribed, "pre-mRNA"
94
mRNAs have ___' cap and a ___' poly (A) tail
5' cap and a 3' poly (A) tail
95
what do the red arrows mean?
different locations where you can have DNA variation
96
from left to right, label the red arrows
- promoter (5' untranslated region)
- exon
- splice site
- intron
- 3' untranslated region
97
_____ are spliced out, ______ remain after splicing
introns, exons
98
5' cap of ____ and ____ for translation initiation
'G' and CH3
99
is the process of splicing 100% accurate 100% of the time?
NO, errors can occur, a retained intron is possible
100
intron
a non-coding region of DNA that doesn't code for a protein in between two regions that do
101
exon
region of DNA that codes for a protein
102
only ~___% of mammalian genome is protein coding
2%
103
in eukaryotes, intron size and number is related to organism ________
complexity
104
in order to have collinearity, introns are spliced out by _________________ in a splicesome
snRNPs (small nuclear ribonucleoproteins)
105
5' splice donor cite ___ (___ in DNA)
GU (GT in DNA)
106
3' splice acceptor site ____
AG
107
what is the purpose of alternative splicing/cleavage?
exons are always placed in order, however, some may be spliced out (or kept in) which affects the type of protein that is made
108
translation
mRNA is used to make a protein
109
how many bases are needed to distinguish 20 AA?
3 (a codon)
110
kmer
short stretch of DNA
111
what equation do we use to determine how many possible combinations of nucleotides there will be?
4^x (x=the number of positions), ex: 4^3 (reading 3 at a time, a codon) = 64 possible combinations
112
mutation introduces _______, which can change the sequence of the codon causing a possible change of the amino acid
variation
113
in general, ____ position mutations have less potential consequences due to redundancy
3rd
114
degenerate
more than one codon for each AA
115
wobble
typically the 3rd base of the codon can vary
116
synonymous
change in DNA sequence does not change AA
117
non-synonymous
change in DNA sequence changes AA
118
nonsense
change in DNA introduces a stop codon
119
start codon, methionine
AUG
120
3 stop codons
UAG, UAA, UGA
121
since the code is read in three's, there is a _________ ______
reading frame
122
each codon specifies same AA in almost all organisms
nearly universal
