1
Q
What was a main research question in the 1950s regarding bacteriophages?
A
In early 1950s, scientists were
wondering why bacteriophages were able to grow on some bacterial strains but were “restricted” from growing on others.
2
Q
What was discovered to answer this question? What do they do?
A
An answer came in 1960s with
discovery of ‘Type I’ restriction
endonucleases.
* These recognize specific DNA
sequences and cleave DNA.
3
Q
What are type I restriction endonucleases?
A
Type I restriction
endonucleases, discovered in
1960s, recognize specific DNA
sequences and then cleave the
DNA sequences…somewhere
else.
4
Q
What do type I restriction endonucleases do?
A
“Restrict” entry of foreign (i.e.,
viral) DNA into bacterial cells.
5
Q
How common are restriction endonucleases?
A
Originally thought to be rare,
later found to be very common.
6
Q
Are type I restriction endonuleases useful?
A
Type I REs are interesting, but
not very useful in molecular
biology
7
Q
How do type I restriction endonucleases work?
A
- Type I restriction endonucleases recognize one area of the DNA
- Then they cut at a downstream site elsewhere
8
Q
What are type II restriction endonucleases?
A
They are known as restriction enzymes and were first reported in 1970.
9
Q
How many restriction enzymes are there?
A
There are thousands that are known now and hundreds commercially available.
10
Q
How do type II REs cleave DNA?
A
‘Type II’ REs cleave DNA
within the recognition site.
- This property has made
them incredibly useful in
molecular biology.
11
Q
DNA restriction sites are…?
A
DNA restriction sites are palindromic! This means they are exact reverse of each other on the top vs. the bottom
12
Q
What is a palindromic sequence?
A
Sequence of nucleotide bases reads the same on the top strand as the sequence of
nucleotide bases reads on the bottom strand of the DNA molecule in 5′ - 3’ direction.
13
Q
Palindromic sequence?
5′-GAATTC-3′
3′-CTTAAG-5′
A
YES
14
Q
Palindromic sequence?
5′-GCACG-3′
3′-CGTGC-5′
A
NO
15
Q
What does DNA cleavage of EcoRI cause?
A
DNA cleavage by EcoRI leaves sticky ends, which are a 4 nucleotide overhang
16
Q
How is the nomenclature of REs derived?
A
Nomenclature of REs is derived from the species (and
‘strain’) name and the order in which they were isolated.
17
Q
Where did we get EcoRI?
A
Escherichi coli
18
Q
Where did we get BamHI?
A
Bacillus amyloliquefaciens
19
Q
Where did we get HindIII?
A
Haemophilus influenzae
20
Q
Why don’t bacterial restriction endonucleases attack the host’s own DNA?
A
The most common reason is that the ‘host’ (bacterial cell)
methylates a base in every copy of the RE site within its own genome.
21
Q
How are DNA sequences that are cut with type II restriction endonucleases rejoined?
A
DNA sequences cut by Type II restriction endonucleases can be rejoined with ligases
22
Q
How comercially available are REs?
A
A huge number of restriction enzymes, covering an almost equally large number of
recognition sequences, are commercially available.
23
Q
What is the sequence and end of EcoRI cuts?
A
EcoRI
G*AATTC
5’ OVERHANG
24
Q
What is the sequence and overhang of HindIII?
A
HinDIII
A*AGCTT
5’ OVERHANG
25
What is the sequence and ends of HpaII?
HpaII C*CGG 5' OVERHANG
26
What is the sequence and ends of HaeIII?
HaeIII GG*CC BLUNT
27
What is the sequence and ends of HindII?
HindII GTY*RAC BLUNT
28
What is the sequence and ends of PstI?
PstI CTGCA*G 3' OVERHANG
29
What is the sequence and end of NotI?
NotI GC*GGCCGC 5' OVERHANG
30
What is gel electrophoresis?
Gel electrophoresis: a method for sorting DNA (& RNA)
sequence fragments by size
31
How are DNA molecules charged at neutral pH?
At neutral pH, DNA
molecules are negatively
charged because of
phosphate groups.
32
Where does DNA tend to move in electrophoresis?
In an electrical field, DNA
will tend to move toward
the positive electrode.
33
Where can we not do electrophoresis? What do we need instead?
Cannot do electrophoresis in a liquid. Need to make a ‘gel’. Most common kind is made from agarose, an uncharged polysaccharide purified from agar of the seaweed, Agar agar.
34
What does the gel contain in addition to agarose and water?
In addition to agarose and water, the gel contains a buffer that provide ions to allow current flow, and to keep the pH slightly above neutral.
35
When was gel electrophoresis invented?
Agarose gel electrophoresis was another extremely important technical innovation of the early 1970s
36
What are the five steps f preparing a gel?
1. The gel tray.
2. Prepare barriers to retain the agarose.
3. Pour molten agarose into the tray.
4. Insert comb to form the wells before the agarose solidifies.
5. Load DNA samples in individual wells and apply voltage.
37
Which molecules go farther in electrophoresis?
longer, larger molecules do not travel as far as the shorter lighter molecules.
- lighter ones will be farther on the gel
38
How do we visualize the DNA?
Size-fractionated DNA is visualized (stained) with a DNA-binding fluorescent dye
39
What is EtBr?
EtBr is an intercalating dye
(between bases)
40
Where do most DNA stains bind?
DNA stains most used now
bind in minor groove.
41
How do we photograph DNA molecules?
Photography of DNA molecules size-fractionated by agarose gel-electrophoresis and stained with ethidium bromide
42
Where do shorter DNA fragments migrate?
Shorter DNA fragments migrate more rapidly through the gel-matrix than longer molecules.
43
What is the migration rate of linear DNA related to?
Migration rate of a linear DNA molecule is inversely related to log of its molecular mass (or # of base-pairs [bp]).
44
What is a standard curve of known DNA used for?
A ‘standard curve’ of known size DNA fragments can be used to extrapolate the size (bp) of an unknown DNA
fragment.
45
What is the1st stage of characterizing a molecule?
Often the 1st stage in the
characterization of an unknown DNA
molecule
46
What 3 factors affect mobility of DNA fragments in the gel?
Several other factors affect mobility of DNA fragments in a gel:
1. Agarose concentration in gel.
2. Topology (physical conformation) of DNA
molecule.
3. Voltage.
47
What can we do to extrapolate bp of unknown DNA?
Based on mobility, can extrapolate the molecular mass (or bp) of an unknown DNA molecule
48
What happens as agarose concentration increases?
As agarose concentration
increases, pore size in
gel matrix decreases.
49
What are smaller pores more resistant to, and what do they favour ?
Smaller pores more resistant to DNA movement, favouring
small DNA fragments, and giving better resolution of size
differences of small fragments
50
What does the concentration of agarose affect?
The concentration of agarose affects the migration of DNA molecules in the agarose gel-matrix
51
How do DNA molecules exist?
DNA molecules can exist in different topologies (conformations): linear,
relaxed circular or supercoiled
52
In what forms can supercoiled DNA be in?
Supercoiled DNA can be either
circular or linear, but the ends of the linear molecule must be
restrained
53
How is DNA typically in cells?
In cells, DNA is often negatively supercoiled.
54
How can we produce positively supercoiled DNA?
Positively supercoiled DNA can
be produced in vitro.
55
Which morphology travels the farthest?
Supercoiled circular DNA travels the fastest through the gel
56
Which morphology travels the medium amount?
Linear DNA travels lest fast than supercoiled but faster than relaxed circular
57
Which morphology travels the slowest?
Relaxed circular DNA travels the slowest through the gel.
58
What happens when voltage speeds up?
Greater voltage speeds up migration rate of DNA fragments during agarose gel-electrophoresis
59
Does %GC influence the migration speed?
The %GC or sequence of a DNA molecule DOES NOT affect migration speed
60
Example of mobility depending on %GC or sequence?
For example, two DNA molecules of equal molecular mass (# ofbase-pairs), one containing 25% GC and the other 75% GC, will migrate through the gel-matrix at the same rate.
61
What is the difference between the 2 types of COVID tests?
There is a cheap and fast way, which is the type of take-home tests we could do.
- The more sensitive and accurate way is through PCR
62
What is the most important technical issue that needs to be overcome for a COVID PCR test to work?
A. Chemical contaminants (inhibitors) that reduce DNA amplification success by interfering with Taq DNA polymerase.
B. Degradation of DNA template by DNAses in nasal mucous.
C. Absence of SARS-CoV-2 genomic DNA in the sample.
D. Sourcing critical materials needed for the test that are in short supply because of global pandemic-related supply chain issues.
A. Chemical contaminants (inhibitors) that reduce DNA amplification success by interfering with Taq DNA polymerase.
63
What are the 5 minimum requirements for DNA synthesis in vitro?
1. A strand of DNA to act as a
template
2. A short, single strand of DNA complementary to part of the template (the ‘primer’)
3. DNA polymerase
4. Deoxyribonucleoside
triphosphates (dNTPs)
5. Mg+ (need by polymerase)
64
What direction dos DNA synthesis proceed in?
DNA synthesis always proceeds in the 3’ direction!
65
Who invented the Polymerase chain reaction?
Polymerase chain Reaction (PCR) invented by
Kary Mullis in 1983, patented in 1985, published
in 1986.
66
What was Mullis' insight?
Mullis’ insight: enzymatic copying of double-
stranded DNA using 2 primers, complementary
to opposite strands could lead to exponential
increase in amount of target sequence.
67
Was the Nobel awarded for PCR? How important is this technique?
led to Nobel Prize and Japan Prize in 1993.
* Has been called the most important technique in
all of molecular biology
68
How much does the amount of DNA change with each cycle?
It doubles each time!! 2 -> 4 -> 8 and so on
69
What temperatures are the DNA cycled through?
PCR requires DNA to be cycled repeatedly through 3 temperatures.
- 92, 60, then 72
70
What are the three steps of PCR?
Step 1: Denature DNA
Step 2: Anneal Primer
Step 3: Primer Extension
71
How many cycles do we generally do for PCR? What does this allow, assuming 100% efficiency?
Generally, ~30-35 cycles.
* This allows (in theory*) for more than a billion-fold
amplification of target DNA
72
At what temperature does denaturation occur?
Temperature: 94-96°C
73
What happens during denaturation?
Double stranded DNA denatures (‘melts’) -> single stranded DNA
74
What temperature does annealing happen at?
Temperature: 50-65°C (dependant on the annealing/denaturation temperature of
primers; Tm)
75
What happens during primer annealing?
Primers bind to their complementary sequences
76
What is Tm dependent on?
Tm is dependent on length and base composition of primers
77
What is Tm for "long" sequences?
For ‘long’ DNA sequences (>100bp) Tm is generally 80-90°C
78
What else does Tm depend on?
Tm also depends on salt concentration of water in which it is dissolved
79
What temperature does elongation/extension occur at?
Temperature: 72°C
80
What happens during elongation/extension?
DNA polymerase binds to the annealed primers and
extends DNA at the 3’ end of the chain
81
What do we use for our temperature cycling?
PCR requires DNA to be cycled REPEATEDLY through 3 temperatures
- We use a thermal cycler "PCR machine" for this
82
What does PCR require?
PCR requires precise temperature cycling
83
How do we accomplish temperature cycling?
Temperature cycling is accomplished using computer-controlled heating/cooling devices (‘thermal cycler’ or ‘PCR machine’)
84
What are the 6 "ingredients" needed for PCR?
* Dinucleoside triphosphates, dATP, dCTP, dGTP, dTTP (‘dNTPs’)
* Mg2+ (essential for enzyme, affects primer annealing)
* Primers (usually, 2)
* Template DNA (need not be pure; can be double or single-stranded)
* Thermostable DNA polymerase (most often Taq, from Thermus aquaticus)
[The ‘original’ version of PCR required addition of DNA polymerase after every
denaturation step!]
* Other ingredients: a salt, Tris (pH control), plus stabilizers
85
What are primers?
Primers = short molecules of singled-stranded DNA (aka
‘oligonucleotides’ or just ‘oligos’ for short), most often 18-25 b long; can be shorter or longer.
86
What gives exponential growth of a product?
Priming between two oligos annealed to opposite strands can give exponential growth of product.
87
What does the size of the product depend on?
Size of PCR product depends on how far apart the annealing sites of the 2 primers.
88
What is the relationship between yield and length of DNA?
PCR products up to 40 kb have been produced but most PCR involves products 2 kb or less. Yield drops with increasing length of DNA product
89
Why are primers usually 18-25 nucleotides long?
Primers that are ~18-25b long are long enough to (usually) match only the intended DNA target sequence.
90
What does successful PCR depend on?
Successful PCR depends on specific binding of primers to the exact positions that will allow us to amplify our ‘target’ DNA.
91
What is specificity of primer binding related to?
Specificity’ of primer binding is related to primer length.
92
What happens if the primers are shorter?
Shorter primers may not be specific enough in their binding; they may match and bind to multiple positions in the genomic DNA, resulting in amplification of ‘incorrect’ (off-target) DNA sequences.
93
What happens if the primers are longer?
Longer primers (>25 b) are more costly but offer little increase in specificity.
94
How can we amplify target sequences for study?
Amplifying target sequences for further study. Amplifying a target sequence from within a
complex mixture (e.g. genomic DNA sample) is ~equivalent to purifying the sequence of
interest. E.g. – could use to amplify a gene sequence for further study in biomedical, or
evolutionary analysis contexts
95
What must we know to design effective primers?
Must know enough about the sequence of interest to design effective primers
96
How can we use PCR to detect rare sequences?
Detection of rare DNA sequences:
Can detect as little as a single copy of DNA sequence, even in a complex mixture.
97
Examples of "rare DNA" applications?
--Detection of--
* bacterial contaminants in food, etc. (e.g., E.coli or Listeria food poisoning)
* bacteria in environmental samples
* pathogens or endosymbionts in organisms (e.g., HIV, COVID-19)
* Forensics – detection of evidentiary DNA at crime scenes
* Environmental DNA (eDNA)
98
What is PCR not good at regarding rare DNA?
But NOT good for determining abundance of these rare sequences!
99
The order of the curve crossing the threshold is proportional to what?
The amplification curves cross the orange line (threshold) in an orderly series determined by starting concentration of
DNA template
100
What is production of DNA limited by in early cycles of PCR?
During early cycles of PCR, production of DNA
product is only limited by the amount in the
previous cycle → exponential growth of
product.
101
What occurs in later cycles of PCR?
In later cycles, dNTPs are less abundant, and
DNA polymerase may start to wear out,
leading to slower growth of product → ‘linear’
phase
102
Eventually, what happens to the growth in amount of product?
Eventually, growth in amount of PCR product
slows down greatly and then stops, as
polymerase and dNTPs start to become
exhausted → ‘plateau’ phase.
103
What does Cp (crossing point, lowest reliable detection) mark?
Cp value marks first point product exceeds
detection threshold of instrument.
104
How does growth appear when looking at a log scale?
When viewed on a log scale, the growth in DNA product during the
exponential phase of PCR appears linear. The log-linear phase provides the
best information to estimate starting amount of DNA (or RNA) template.
105
What is the number of cycles to reach Cp a measure of?
The number of cycles to reach Cp is also a measure of starting DNA template
amount.
106
What do we use to determine growth?
Growth in amount of PCR product is monitored by using a reporter dye, and a
PCR machine capable of detecting fluorescence in each well.
107
What dye do we use normally?
SYBR Green is simplest and cheapest reporter dye.
108
When does SYBR fluoresce the most?
SYBR fluoresces much more strongly when bound to double-stranded DNA.
109
What does SYBR bind?
Binds primarily to minor groove in double-stranded DNA.
110
What other methods can we use to track PCR progress?
Other methods of
tracking progress of
qPCR use special
fluorescent probes that
come in different
colours.
111
Can we track multiple reactions in the same well?
Can track multiple
reactions (different
targets) in same
reaction well, using a
different colour for
each target.
112
How many samples can be run at once?
Can run 96 or 384
samples at once.
113
What can we quantify with qPCR?
Quantify amount of starting DNA of a particular sequence.
* E.g. How abundant is a particular type of bacteria (or virus!) in a sample.
114
How do we measure the rate at which a gene is transcribed?
Measuring rate at which a particular gene is transcribed.
* Need to convert mRNA to cDNA first.
* Use reverse transcriptase for this.
115
4 steps of conversion of RNA to cDNA?
1. Oligo dT primer is bound to mRNA
2. Reverse transcriptase
(RT) copies first cDNA strand
3. Reverse transcriptase
digests and displaces mRNA
and copies second strand of
cDNA
4. Double strand cDNA made
116
4 steps of PCR?
A. Double strand DNA
B. Denature
C. Anneal primers
D. Polymerase binds
117
What is the amount of PCR product proportional to?
The amount of PCR product (in exponential phase of qPCR)
is proportional to starting amount of DNA
118
C
119
How common is the Sanger dideoxy chain terminating method?
One of two methods of
sequencing DNA invented in
1970s.
* Still used today.
* Remains gold standard for
accuracy and convenience for
sequencing small numbers of
samples
120
In what direction does synthesis of a new strand proceed?
DNA synthesis of the new strand always proceeds in the 5’ to 3’ direction!`
121
What is released during the formation of a phosphodiester bond?
FORMATION OF A PHOSPHODIESTER BOND CATALYZED BY DNA POLYMERASE (i.e. addition of a base to a growing DNA strand)
- P-P is released from the dNTP during the reaction.
122
What type of reaction is this?
a condensation reaction
123
What terminates DNA synthesis?
Dideoxyribonucleoside triphosphates terminate DNA synthesis
124
What do we look with the dideoxy reaction?
Consider what would happen in a DNA extension (synthesis) reaction, in which most of the dGTP is regular dGTP,
but a small amount, say 5%, is ddGTP.
125
What is true in this case for the addition of nucleotides?
In that case, most (95%) of the time when ‘G’ is incorporated, it would be a normal dGTP, and strand elongation
past that base could continue. BUT, 5% of the time, a ddGTP would be incorporated, and when that happened,
there would be no further extension of that particular DNA strand.
126
What does this give us for daughter strands?
This would give us DNA daughter strands of varying lengths, the lengths of which are determined by where the ‘G’s occur in the sequence.
127
What else can we look at in other cases?
We could do the same thing for the
other bases: ‘Spike’ the DNA
polymerization cocktail with small
amounts of ddATP, ddCTP, and ddTTP
(in addition to the ddGTP).
128
What would we get in this case?
In this case, we would get a subset of
DNA elongation products terminating
with a ddNTP base at every position in
the DNA sequence.
129
2 issues with this?
BUT – (1) how do we keep track of
which bases are terminating which
fragments?
* AND – (2) how do we sort out the
different fragments by size?
130
What solves our problem of tracking?
(1) We attach (different) fluorescent colours to each type of ddNTP (
e.g., blue, red, ‘black’, green in this example)
131
Solution to our second problem of sorting?
(2) We use gel electrophoresis to sort the fragments by size. The smallest fragments
will represent DNA sequences terminating close to the primer.
132
How is fluorescent dideoxy sequencing?
It is usually automated
133
What does Gel electrophoresis do to separate ssDNA?
Gel electrophoresis uses denaturing
polyacrylamide gel (contains urea) to separate
single-stranded DNA fragments by size. This type
of gel gives very fine resolution, ability to
distinguish fragments that differ by 1 base in size.
134
What happens to ddNTP terminated fragments?
As ddNTP-terminated fragments migrate in the
gel, they pass a laser beam, that excites the
fluorescent dyes, and a camera that records the
flash of coloured light that results.
135
What is the main drawback of dideoxy sequencing?
Sanger dideoxy sequencing
accurate but SLOW
* Originally, 4/tubes/sequence
136
How fast is the automated sequencing?
Later, automated
fluorescent sequencing
much faster, but still only
~100 samples/day
(~650b/seq) -> ~65,000
bases/day/machine.
137
What are the pros of Sanger dideoxy sequencing?
* Very accurate
* Relatively long sequencing reads (up to nearly 1,000b; although ~650b more common)
* Easy to do; can be automated.
* Low cost (for small numbers ofcsamples).
* Continues to be used for all these reasons.
138
What are the cons of Sanger dideoxy sequencing?
* Too slow for many applications, such as genome sequencing!
* Costly when scaled up to acquire lots of data.
* Requires purification and
preparation of each individual DNA sequence that is being studied.
* These limitations led to invention of other methods, so called ‘next-generation’ methods.
139
How much did the human genome project cost?
The Human Genome Project cost ~$3 billion (U.S.)!!
- Sequencing the first human
genome was incredibly
expensive.
140
Why was the human genome project so expensive?
Many reasons, but biggest
reason is that Sanger dideoxy
sequencing is too slow and
costly
141
How much does a genome sequence cost now?
A human genome sequence now costs <<$1,000.
* What changed?
142
What was the capacity back then (bases/day)?
Human Genome Project
Consortium in 2000 (total of
many labs in 6 countries): 8.64 x 107 bases/day (~~108
bases/day)
143
What was the capacity in the Bentzen lab?
Bentzen lab Illumina MiSeq DNA sequencer: ~1.5 x 10^10
bases/day
144
Automating helped, but left what issue to persist?
Automating the process helped but wasn’t enough.
- Each ‘sample’ needs to be purified to consist of only 1 DNA single sequence.
145
Why can't we use individual samples for genome sequencing?
Genomes are big!
* Handling and sequencing individual samples
(DNA templates) is too slow for genome sequencing
146
What approach was developed?
An approach was needed that allow for many (millions) of DNA segments to be sequenced at once ( = ‘massively parallel’).
- these are considered "next generation sequencing methods"
147
What are the 3 next generation sequencing methods?
* Pyrosequencing
* Illumina
* Ion Torrent
-> Use variety of technologies, but all sequencing
by synthesis
148
What is important for Illumina DNA sequencing?
DNA needs to be short segments: accomplished by shearing or use of
short PCR products.
149
How are adaptor sequences added to the ends of DNA?
‘Adaptor’ sequences are added by ligation to ends of DNA segments.
150
How are the DNA segments arrayed?
DNA segments to sequenced are randomly arrayed across flow cell surface.
151
How do we amplify individual molecules into clusters?
Bridge amplification’ used to amplify single DNA molecules into clusters of identical DNA molecules.
152
What do adaptors add?
Adaptors add sites for attachment of DNA sequencing primers and
enable attachment to the oligonucleotides on the surface of the flow
cell.
153
How does illumina sequencing occur?
Sequencing occurs by addition of fluorescently labeled nucleotide analogs, 1 based at a time. These dNTP analogs are chain terminators (like Sanger) but are reversible (unlike terminators used in Sanger sequencing), so that after chemical treatment of the newly added dNTP, the chain can continue to elongate.
154
What occurs after each dNTP is added?
After each dNTP is added, sequencer pauses and exposes flow cell to laser and takes a picture to record (for each DNA cluster) what base
was incorporated in each cluster. Process continues for a few hundred cycles.
155
How does the computer interpret the data?
Computer interprets the data to infer the DNA sequence within each DNA cluster on the flow cell.
156
How many distinct DNA sequences can be done this way?
Millions of distinct DNA sequences determined simultaneously this
way (= massively parallel DNA sequencing)
157
What is unique about nanopore sequencing?
NOT DNA sequencing by synthesis.
* Single molecule at a time (no pre- amplification by PCR).
158
How is DNA isolated?
Enzyme unwinds DNA; a single
strand is pulled by an electrical
current through a pore in a
membrane.
159
What does each base produce?
Each base produces a
characteristic disturbance in
electrical current, which can be
used to read the base as it travels through the pore.
160
What are the pros of nanopore?
* Long reads - up to 100kb!
* No amplification step to boost amount of template DNA before sequencing.
* Small, highly portable DNA sequencer connects to USB port on a computer.
* Can be used in the field to get rapid results.
* Can detect methylated bases.
161
What are the cons of nanopore?
Cons:
* Slightly less accurate than other methods.
* Other long read, single DNA molecule sequencing technologies exist.
162
Sanger, Illumina, Nanopore: massively parallel?
Sanger: NO
Illumina: YES
Nanopore: YES
163
Sanger, Illumina, Nanopore: sequencing by synthesis?
Sanger: YES
Illumina: YES
Nanopore: NO
164
Sanger, illumina, Nanopore: single molecule?
Sanger: NO
Illumina: NO
Nanopore: YES
165
Sanger, Illumina, nanopore: chain termination?
Sanger: YES, ireversible
Illumina: YES, reversible
Nanopore: NO
166
Sanger, Illumina, Nanopore: accuracy?
Sanger: 99.99%
Illumina: up to 99.9%
Nanopore: up to 98->99%
167
Sanger, illumina, nanopore: read length?
Sanger: 650-1000
Illumina: 75-600
Nanopore: 100kb
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