Quiz 2: Lecture 7 Videos Flashcards
Sanger Sequencing video:
The first method of sequencing the genetic code was devised by…
Fred Sanger
What must be done to sequence the DNA?
It must first be separated into 2 strands
The strand to be sequenced is copied using chemically altered bases
These altered bases cause the copying process to stop each time one particular letter is incorporated into the growing DNA chain
This process is carried out for all 4 bases, and then the fragments are put together like a jigsaw to reveal the sequence of the original piece of DNA
The Pyrosequencing Reaction Cascade System video:
What does pyrosequencing technology capitalize on?
Elegant chemistry to deliver real-time quantitative sequence data
The Pyrosequencing Reaction Cascade System video:
What do 4 enzymes do? What happens?
4 enzymes convert the synthesis of a DNA molecule into a pattern of light signals
DNA elongation catalyzed by DNA polymerase is accompanied by the release of pyrophosphate
ATP sulfurylase converts Pyrophosphate to ATP
ATP powers oxidation of luciferin by Luciferase which generates a light signal recorded as a pyrogram peak
Finally, Apyrase removes any unincorporated nucleotides remaining in the reaction
What is the light signal proportional to?
The amount of nucleotides (added) incorporated into the synthesized DNA strand
What happens when 3 nucleotides are incorporated by polymerase?
3 molecules of pyrophosphate are generated (ATPs are generated)
The 3 pyrophosphate molecules are converted to 3 ATP molecules and these power an equimolar oxidation of luciferin
The result is a recorded Triple peak
What happens to the remaining unincorporated nucleotides?
They are removed from the reaction in preparation of the next dispensation of nucleotides
Nucleotides are added to the reaction in what order?
If during a dispensation, no nucleotides are incorporated…
Predefined dispensation order
No pyrophosphate is generated (No incorporation, No light signal) and consequently no light signal is recorded
These blank nucleotide dispensations resulting in no signal can be used as what?
Built in negative sequencing controls
What happens in the pyrosequencing reaction cascade system?
Another dispensation of nucleotides takes place, pyrophosphate is generated, then converted into ATP, and a corresponding light signal results from luciferin oxidation, in this case a Double Peak
In this manner the pattern of light signals recorded in a pyrogram is..
An easily interpreted representation of the true nucleotide sequence of the analyzed DNA
Chromatography Animation (IQOG-CSIC) video:
What is shown in the video?
Stationary phase > Mixture of products on top right > Is moved toward left side by vehicle > Put inside stationary phase > Mobile phase on left pushes it to right > It goes down from pipe on right side into vehicle in Separate colors (Red, Yellow, Blue) with Black balls remaining inside Stationary Phase
Separated products: Red, Yellow, Blue balls
How nanopore sequencing works video:
What is shown in this video?
Flow cell
DNA (or RNA), Motor protein, Adapter sequence
2048 membrane wells, each containing a nanopore
Tether, Nanopore
Disruption of ionic current, measured in signal trace - 400 bases per sec
Sequenced strands
(Used in over 80 countries and in space)
Illumina sequencing technology video:
What are the steps?
Sample Prep, Cluster Generation, Sequencing, Data Analysis
Illumina sequencing technology video:
Sample Prep
What is the first step?
What happens during it?
Sample preparation begins with extracted and purified DNA
The first step in Nextera sample preparation is Tagmentation
During Tagmentation, Transposomes (on both sides of double stranded DNA) simultaneously fragment and tag the input DNA with adapters
Once the adapters have been ligated…
Reduced cycle amplification adds additional motifs (Adapters on both sides of DNA insert) (Primers (2) and DNA insert)
such as Sequencing primer binding site 1, Sequencing primer binding site 2 on both sides of DNA insert, Region complimentary to flow cell oligo (left), Region same as flow cell oligo (right), Index 2 (left) and Index 1 (right)
Cluster Generation:
Clustering is a process where each fragment is isothermally amplified
The flow cell is a glass slide with lane, each lane is a channel coded with a lawn composed of 2 types of oligos
Hybridization is enabled by what?
(The flowcell contains two types of oligos)
Hybridization is enabled by the first of the two types of oligos on the surface
This oligo is complimentary to the adapter region on one of the fragment strands
What does a polymerase create?
What is the double stranded molecule?
A complement of the hybridized fragment
(Region complementary to flow cell oligo (top), Reverse strand (under))
The double stranded molecule is denatured, and the original template is washed away
How are the strands amplified?
What happens in this process?
Clonally amplified, through bridge amplification
The strand folds over and the adapter region hybridizes to the second type of oligo on the flow cell
What do polymerases generate?
What is bridge amplification?
The complimentary strand, forming a double stranded bridge
This bridge is denatured, resulting in 2 single stranded copies of the molecule that are tethered to the flow cell (Forward strand, Reverse strand)
The process is repeated over and over, and occurs simultaneously for millions of clusters resulting in Clonal amplification of all the fragments
What is after bridge amplification?
What happens to the 3’ prime ends?
The reverse strands are cleaved and washed off, leaving only the forward strands
The 3’ prime ends are blocked to prevent unwanted priming
Sequencing:
What does sequencing begin with?
Extension of the first sequencing primer to produce the first read (Sequencing primer, Template)
With each cycle, 4 fluorescently tagged nucleotides compete for addition to the growing chain
Only one is incorporated, based on the sequence of the template (Flow cell, Template)
After the addition of each nucleotide, the clusters are excited by a light source and a characteristic fluorescent signal is emitted
This proprietary process (Sequencing) is called…
What determines the length of the read?
What determine the base call? (2)
Sequence by synthesis (Flow cell, C G A A T T)
The number of cycles determines the length of the read
The emission wavelength along with the signal intensity determine the base call
For a given cluster, all identical strands are read how?
Simultaneously
Hundreds of millions of clusters are sequenced in a massively parallel process
(This image represents a small fraction of the Flow Cell)
What happens after the completion of the first read?
The read product is washed away
In this step, the Index 1 Read Primer is introduced and hybridized to the template
(Index 1 Primer, Flow cell)
The read is generated, similar to the first read
What happens after completion of the index read?
What does the template now do?
How is Index 2 read?
The read product is washed off and the 3’ prime end of the template is deprotected
The template now folds over and binds the second oligo on the Flow cell
Index 2 is read in the same manner as Index 1
Index 2 Read product is washed off at the completion of this step
Polymerases extend the second flow cell oligo, forming what?
A double stranded bridge
This double stranded DNA is then linearized and the 3’ prime end is blocked
(Forward strand, Reverse strand- Paired-end Sequencing)
The original forward strand is cleaved off and washed away, leaving the reverse strand
What does Read 2 begin with?
Introduction of the Read 2 sequencing primer (Read 2 primer)
As with Read 1, the sequencing steps are repeated until the desired read length is achieved
The Read 2 product is washed away
(This entire process generates billions of reads representing all the fragments)
Data Analysis:
Sequences from pooled sample libraries are Samoa rated based on what?
Unique indices introduced during the sample preparation
(Similar sequences)
For each sample, reads with similar stretches of base calls are what?
Forward and reverse reads are what?
What is the paired in information used to resolve?
Locally clustered
(Local sequence clustering)
Paired, creating continuous sequences
These contiguous sequences are aligned back to the Reference genome for variant identification
Ambiguous alignments
Maxam and Gilbert DNA Sequencing:
What are the steps? (4)
DNA Preparation, Chemical Treatment, Electrophoresis, Autoradiography
Maxam and Gilbert DNA Sequencing:
DNA denaturation:
At 95 degrees Celsius
Sequencing of 5’ to 3’ DNA is left
Maxam and Gilbert DNA Sequencing:
DNA radioactive labeling:
32^P in each tube of DNA
DNA labeled on the 5’ end, with 32^P
Maxam and Gilbert DNA Sequencing:
Chemical Treatment:
What does it do?
What does this lead to the formation of?
Formic Acid
Formic acid: Breaks the link between a purine (A or G) and the deoxyribose to which it is attached
The formation of Apurinic site, Different Apurinic sites
First tube- A+G
Maxam and Gilbert DNA Sequencing:
Dimethyl Sulfate:
Methylation of Guanines by dimethyl sulfate
Removal of modified G from polynucleotide chain
Second tube: G
Maxam and Gilbert DNA Sequencing
Hydrazine:
The pyrimidines (C+T) are hydrolysed using hydrazine
Third tube: T+C
Maxam and Gilbert DNA Sequencing
Sodium chloride:
The addition of sodium chloride to the hydrazine reaction inhibits the reaction of thymine for the C-only reaction
Fourth tube: C
Maxam and Gilbert DNA Sequencing
Piperidine:
The modified DNAs cleaved by hot Piperidine at the position of the modified base
(Put into all 4 tubes)
(Cuts the DNA strands?)
Maxam and Gilbert DNA Sequencing:
Acrylamide Gel electrophoresis:
(Put 4 tubes into the wells, Plug it in, watch DNA strands move)
Movement of DNA in Gel Electrophoresis (2)
- The negative charge of its phosphate backbone moves the DNA towards the positively charged anode
- Smaller DNA molecules migrate more rapidly than larger fragments
What are the DNA bands visualized by in Gel electrophoresis?
X-ray film
Overnight exposure, Transfer of the DNA fragments to the X-ray film
What are the 4 wells of the Gel electrophoresis?
A+G, G, T+C, C
