Week 3 (PCR and RNA Review) Flashcards
PCR
Polymerase Chain Reaction
what steps are involved to prepare DNA for replication?
- 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
why is an RNA primer so important?
it makes a FREE 3’ hydroxyl available for DNA polymerase to bind
what adds a free 3’ hydroxyl group so that DNA polymerase can bind?
primer
In which direction is DNA synthesized?
5’ to 3’
on a forward facing DNA strand (5’—- 3’), where would the primer need to be placed?
toward the 3’ end because DNA is read 3’ to 5’ so that it can be synthesized 5’ to 3’
on a reverse facing DNA strand (3’—- 5’), where would the primer need to be placed?
toward the 3’ end because DNA is read 3’ to 5’ so that it can be synthesized 5’ to 3’
when will DNA polymerase stop reading the DNA strand?
when there is no longer a template to read from
requirements for replicating DNA
- DNA template
- DNA polymerase
- nucleotides (dNTP)
- primers
what is the equation for number of copies of target?
- N*2^C
- N = number of starting copies
- C = number of cycles
N*2^C
number of copies of target strand
N*2^C - what does N mean?
number of starting copies
N*2^C - what does C mean?
number of cycles
ex: N*2^C, you have 1 starting copy and 2 cycles
4
ex: N*2^C, you have 1 starting copy and 25 cycles
33.5M
Why do you lose some DNA every time you replicate?
the primer attaches to the DNA template and will start copying at your target DNA, it is only trying to amplify your target DNA
What are the 3 steps in PCR?
- denaturation
- annealing
- extension
what occurs during the first step of PCR?
- denaturation
- 94 degrees Celsius
- melt the two strands apart
what temperature does it need to be for denaturation?
94 degrees C
why do we heat up the DNA to separate the 2 strands?
weak hydrogen bonds hold them together and so they can be separated by heat
what can be used to separate the DNA from the histones?
phenol
what occurs during the second step of PCR?
- 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
what temperature does it need to be for annealing?
54 degrees Celsius
why does it need to be 55 degrees Celsius during the annealing process?
warm enough to keep DNA from coming back together but cool enough to have primers bind to each single strand of DNA
why does the temperature need to cool down rapidly between denaturation and annealing?
if it is done slowly, you will have nonspecific priming
what occurs during the third step of PCR?
- extension
- 72 degrees Celsius
- optimal temperature for DNA polymerase
- extend in the 5’ to 3’ direction
what temperature does it need to be for extension?
72 degrees celsius
why does it need to be 72 degrees celsius during extension?
optimal temperature for DNA polymerase
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
what are the 3 things that are key to PCR success?
- primer specificity
- annealing temp
- Mg2+ concentration
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)
what is the k in 4^k?
the number of bases in the primer
for primer specificity, what does it mean if k=4?
there are 4 bases in the primer, so there is 1 in 256 bases
how does annealing temperature affect specificity (low vs high temp)?
- low temp = less specific
- high temp = more specific
how does Mg2++ concentration affect specificity (low vs high concentration)?
- low Mg = higher specificity
- high MG = lower specificity
what is the minimum primer length to assume that it only binds to one location in the genome?
between 15 and 17
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
What would a really good PCR look like?
distinct bands
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
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
how do you solve what the optimal PCR is? You have 2 variables.
Hold one variable constant and change the other
when finding the optimal PCR, how can you speed up the process?
using multiple thermal cyclers
do you have to optimize for every primer?
yes
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
what primer can you add, knowing you have an RNA with a Poly A tail?
an oligo dT primer
What is needed for a polymerase to bind to the molecule?
free 3’ hydroxyl
what molecule do we need to read RNA and make a new strand of DNA?
reverse transcriptase
RT-PCR
Reverse Transcriptase Polymerase Chain Reaction
what is the purpose of RT-PRC?
synthesizing a new strand of DNA from mRNA
some limitations of PCR
- size
- base complexity
- secondary structure
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
in general using specialized polymerases one can get about _____ kb
30
why does base complexity limit PCR?
repetitive sequences are a problem for primer specificity
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
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
examples of a secondary structure and its shape
a secondary structure of RNA
tRNAs, clover leaf
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)
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.
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
in sanger sequencing, what does each of the 4 tubes contain?
- DNA fragment to be sequenced
- primer
- dNTP (bases A, G, T, C)
- polymerase
- ddNTP
ddNTP
dideoxynucleotide triphosphate
what is a terminator?
- ddNTP
- when DNA polymerases use the ddNTP the process stops
there is a _____ ratio of ddNTP to dNTP so you don’t stop at only the first point they appear
low
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)
what did the Human Genome Project use to sequence the human genome?
dideoxy-sequencing method
dideoxy-sequencing method can be _____________, meaning humans spend less time analyzing the results
automated
central dogma
DNA–>RNA–>Protein
where does transcription occur in the cell?
the nucleus
where does translation occur in the cell?
the cytoplasm
key components needed for transcription
- a DNA template
- the raw materials (ribonucleotide triphosphates - A,C,G,U) needed to build a new RNA molecule
- the transcription apparatus, consisting of the proteins necessary for catalyzing the synthesis of RNA
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
why is the nontemplate strand of DNA not usually transcribed?
it would make a different protein, the strand is complementary
RNA is synthesized in the ____’ to ___’ direction
3’ to 5’
during transcription, DNA is read in the ___’ to ___’ direction
3’ to 5’
what are two differences between DNA replication and transcription?
- incoming nucleotides have and OH at 2’
- RNA uses Uracil
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)
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.
the number of genes ________ strongly correlated with organismal complexity
IS NOT
T2T
telomere to telomere
when was the first genome read telomere to telomere, when was the human genome project really finished?
2022
a lot of what is transcribed and not translated can have a lot of ____________ roles
regulatory
genes can be turned on and off depending on:
- time in development
- nutrients
- stress
what do regulatory regions of a gene determine?
if transcription occurs: the what, when, where, how much
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)
what are control structures in transcription?
- regulatory promoters
- core promoters
- transcription factors
- enhancers
regulatory promoters
- upstream of the core promoter
- transcription factors can bind here
- affect the RATE of transcription
regulatory promoter affect the _____ of transcription
RATE
enhancers
distal locations can also enhance transcription
core promoter
where polymerase binds
what is upstream on the gene?
before the promoter
what is downstream on the gene?
after the promoter
_____ is the fundamental unit of heredity
gene
if the promoter/enhancers/regulatory proteins “say so”, a protein-coding gene is ____________, and the result is a “___________”
transcribed, “pre-mRNA”
mRNAs have ___’ cap and a ___’ poly (A) tail
5’ cap and a 3’ poly (A) tail
what do the red arrows mean?
different locations where you can have DNA variation
from left to right, label the red arrows
- promoter (5’ untranslated region)
- exon
- splice site
- intron
- 3’ untranslated region
_____ are spliced out, ______ remain after splicing
introns, exons
5’ cap of ____ and ____ for translation initiation
‘G’ and CH3
is the process of splicing 100% accurate 100% of the time?
NO, errors can occur, a retained intron is possible
intron
a non-coding region of DNA that doesn’t code for a protein in between two regions that do
exon
region of DNA that codes for a protein
only ~___% of mammalian genome is protein coding
2%
in eukaryotes, intron size and number is related to organism ________
complexity
in order to have collinearity, introns are spliced out by _________________ in a splicesome
snRNPs (small nuclear ribonucleoproteins)
5’ splice donor cite ___ (___ in DNA)
GU (GT in DNA)
3’ splice acceptor site ____
AG
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
translation
mRNA is used to make a protein
how many bases are needed to distinguish 20 AA?
3 (a codon)
kmer
short stretch of DNA
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
mutation introduces _______, which can change the sequence of the codon causing a possible change of the amino acid
variation
in general, ____ position mutations have less potential consequences due to redundancy
3rd
degenerate
more than one codon for each AA
wobble
typically the 3rd base of the codon can vary
synonymous
change in DNA sequence does not change AA
non-synonymous
change in DNA sequence changes AA
nonsense
change in DNA introduces a stop codon
start codon, methionine
AUG
3 stop codons
UAG, UAA, UGA
since the code is read in three’s, there is a _________ ______
reading frame
each codon specifies same AA in almost all organisms
nearly universal
