Midterm Flashcards
DNA synthesis reaction requirements
- DNA template - purpose is because DNA polymerase requires a template so that DNA replication can occur)
- Primer/3’ OH = The primer is a single stranded DNA with an OH located on the 3’ end and will make base pairs for the template and provides the 3’ OH end for DNA polymerization
- dNTPs - nucleotides to basepair with the template and be incorporated by DNA polymerase
- DNA polymerase - used to synthesize a new DNA strand
FRED Sanger
developed the Sanger sequencing method (also known as chain-termination sequencing), which enabled scientists to determine the exact sequence of nucleotides in DNA
What is a ddNTP and how is it used in Sanger-based sequencing?
-prevents DNA synthesis and allows for chain termination
-This is a nucleotide that has an 3’ “H” on its ribose sugar instead of an “OH” and because of that, when it’s incorporated into a growing chain, the absence of the 3’ OH means that nothing else can be added (H is unable to act as a nucleophile in phosphodiester bond formation and so the bond between the alpha and beta phosphate group wouldn’t be able to break and allow for the inorganic phosphate leaving group)
In the original Sanger sequencing method where, why and how was the primer end-labeled with P32 ?
- Labels are added onto primers at the 5’ end
- Primers, also known as oligonucleotides, have a 5’ with an OH attached so that it’s easier to attach a radioactive label which is a phosphate group onto it
-T4 polynucleotide kinase takes the gamma phosphate (32 P) from ATP and adds it to the 5’ OH end of the primer and this is what becomes the label
In a lane on a polyacrylamide sequencing gel (PAGE) what are the differences between each
successive fragment ?
-each fragment product inputted from the tubes ends at a different point due to the incorporation of chain-terminating nucleotides.
- The gel separates these fragments by size, allowing the sequence to be read base by base from the smallest to the largest fragment, corresponding to the DNA sequence from the 5′ to 3′ direction.
PCR-based Cycle Sequencing
- Iterated cycles of denaturation and DNA synthesis to amplify a piece of DNA using 2 primers targeted in a region of DNA
- you take a piece of a dsDNA fragment
-Denature it at 94 degrees Celsius so that it becomes 2 single strands - you cool the strands to 50-60 degrees celsius so that the each of the primers can be hybridized to each of the strands
- raise the temperature to 72 degrees celsius so that DNA polymerase would be able to extend polymerization downstream
-repeat
Why does PCR-based cycle sequencing only require one primer?
-This is so that the amplification of product is linear and not exponential
- basically you’re only amplifying one fragment multiple times at a time
What are the qualities that make a good DNA polymerase for chain-termination sequencing?
- High processivity because this means that DNA polymerase will have an increased affinity to attach to the template and produce a long chain so it doesn’t detach before a ddNTP can terminate the chain.
- Negligible 5’ to 3’ Exonuclease activity so that the DNA polymerase is not cleaving the 5’ ends of newly synthesized strands which would alter the sequence
- Negligible 3’ to 5` exonuclease activity so that the DNA polymerase is not cleaving the 3’ ends of newly synthesized strands which would alter the sequence
What is the difference between a forward, reverse, universal and internal primer with respect to DNA sequencing ?
-Forward primer = Primer that is located on the template strand of DNA (3’ to 5’) and will provide a forward sequence
- reverse primer = Primer that is located on the 3’ to 5’ end of the non-template strand of DNA and will provide a reverse sequence
- Internal primers = A primer that is designed to anneal at a position within the sequence of two fragments of DNA and so it will make up the sequences between so that the fragments can become one sequence
-Universal primer = Primers with a known sequence that is complementary to nucleotide sequences that are very common in a particular set of DNA molecules and cloning vectors. Thus, they are able to bind to a wide variety of DNA templates.
What is the DNA polymerase that is most often used?
- Taq polymerase
Explain why pUC19 was vector of choice for DNA sequencing projects in the Sanger Era
- Because you didn’t need to know the sequence of the inserted DNA into the plasmid
- The sequence of the plasmid is known and the sequence of the M13 forward and reverse primers are known and so when DNA is cloned into one of the multiple cloning sites, a sequencing reaction can be performed with both primers and so you can get the forward and reverse sequences of whatever was cloned into the multiple cloning sites
The read length of Sanger Sequencing is ~700bp – how does that property lead to the shotgun sequencing strategy of genomes
The ~700 bp read length of Sanger sequencing limits how much of the genome can be read in a single sequence, especially for large genomes.
To tackle this, the shotgun sequencing strategy is used: the genome is randomly fragmented into many smaller pieces that fall within this read length.
Each fragment is then individually sequenced, and computational methods are used to align overlapping sequences and reconstruct the entire genome.
This approach allows comprehensive sequencing despite the limited read length of each individual Sanger sequence.
What is the importance of molecular cloning to Sanger Sequencing of Genomes ?
- cloning enables the insertion of genomic DNA fragments into vectors (like plasmids), which can then be grown and quickly replicated in bacterial cells. This breaks the genome into manageable pieces that can each be sequenced individually.
- Cloning provides many identical copies of each DNA fragment, ensuring enough template for high-quality sequencing. This is crucial in Sanger sequencing, which requires sufficient template DNA for reliable detection of each nucleotide.
What is sequence coverage ?
The amount of times that regions of the genome have been sequenced
What are the strengths and limitations of Sanger based chain termination sequencing
- easy to automate
-Automated sequencers with multiple capillary gels working in parallel can read up to 384 different sequences in
one hour
-Almost 7MB can be obtained in 24 hours - sequencing a genome can be performed in a relatively short time
- at least 5x sequence depth or coverage is needed for increased accuracy
-realized that progress in Genomics would be slow if had to rely on
Chain-Termination Sequencing technology
Kary Mullis
invented the Polymerase Chain Reaction (PCR) technique, which allows for the rapid amplification of specific DNA segments.
How are Next Generation Sequencing methods massively parallel?
- Next-generation sequencing (NGS) methods are massively parallel because they sequence millions of DNA fragments simultaneously in a single run.
- This is achieved by using sonication to randomly break the DNA into numerous fragments (100bp - 500bp long), anchoring them to a solid surface, and then performing sequencing reactions on all fragments at the same time thus allowing for the rapid generation of massive amounts of sequence information
What are adaptors and what are their importance to Next Generation Sequencing ?
-short pieces of dsDNA that have a known sequence and are ligated to the randomly sonicated template DNA library ( DNA fragments) and their sequence matches the oligonucleotides that are immobilized on the slide
- Then the pieces of ligated DNA are denatured and the resulting single stranded molecules base pair with the immobilized oligonucleotides on the slide
Why is DNA amplification an important step in Next Generation Sequencing ?
DNA amplification is crucial in Next Generation Sequencing (NGS) because it generates multiple copies of each DNA fragment, amplifying the signal for accurate detection
Immobilization of DNA fragments in a sequencing library by base pairing to oligonucleotides on a glass slide
-There’s a flow cell that looks like a glass slide with a coating of oligoucleotides/primers that all have the same sequence
- the sequence of the oligonucleotides matches that of the adaptors that have been added to the template DNA fragment
- since the adaptors match the sequence of oligonucleotides in the well, they will base pair to form an attachment
-adaptors will be added in the solution of genomic DNA that’s been sonically fragmented in the wells
Immobilization of DNA fragments by base pairing to metallic beads
- the DNA preparation involves the genome of the organism being sequenced to be sonically fragmented with adaptors ligated onto the ends with a known sequence
- The adaptors have a biotin label attached to the 5’ end which then gets added to the DNA fragment when they are ligated onto them
- each fragment get attached to one bead so one bead with one template per emulsion and then ultimately the beads get transferred to a solid support with one droplet of the emulsion per well and one template per well
What are the drawbacks of nanopore sequencing currently ?
relatively high error rate (~15%) but each year it
decreases as the technology develops
What is the read length of the nanopore sequencing platform
Extremely Long Read Lengths up to 100s kb
What is a CsgG nanopore?
The protein that forms a nanopore that allows the nucleic acids from the dsDNA strand that gets unwound by helicase to be driven through the pore so that each nucleotide can be determined in the sequence
Explain how nanopore sequencing works
- Use synthetic membrane with small pores just large enough for DNA to pass through
- Electrical current set up so electrophoresis causes the DNA to approach the nanopore
- Helicase in vicinity of nanopore unwinds dsDNA so only one strand passes through
- Sequence of the strand can be read because each of the 4 nucleotides has a different shape and therefore modifies the nanopore in a different manner resulting in a slightly differentdisruption of the flow of ions passing through the membrane
- The disruptions are measured to deduce
sequence of the ssDNA molecule
How does nanopore sequencing technology of Oxford Nanopore technologies fulfill criteria of
fourth generation ?
It doesn’t require DNA polymerase, primers or added nucleotides
What is Interpulse Duration (IPD) in SMRT technology and what is its importance to genomics?
- Measures the time between incorporations of the correct nucleotide that will pair to the template strand
- As nucleotides are incorporated, they release a fluorescent signal when it reaches the active site of the polymerase and IPD can detect this time between each incorporation
-This is important for genomics because it can
detect epigenetic modifications such as methylation on a DNA template
Is SMRT massively parallel ?
- Yes it is
-Zero-Mode Waveguides (ZMWs) will be present on a flow cell with 10s of thousands per slide with a single DNA polymerase at the bottom of a ZMW and a single molecule of DNA
as a template there as well - Each ZMW contains a single DNA polymerase molecule that synthesizes DNA in real-time, capturing fluorescent signals from nucleotide incorporations.
- This parallel approach allows SMRT to rapidly generate long-read sequences across many DNA molecules simultaneously
What is the read length of PacBio platform ?
> 10,000bp currently
What is the PacBio CCS technology?
PacBio’s Circular Consensus Sequencing (CCS) technology is a method that enables highly accurate single-molecule sequencing.
- In CCS, a DNA molecule is circularized and read multiple times by the sequencing polymerase within a single Zero-Mode Waveguide (ZMW).
- By sequencing the same molecule repeatedly, CCS generates multiple reads for each DNA strand, allowing for the correction of random errors and thus achieving high accuracy
In SMRT sequencing the fluorescent dye is attached to the phosphate of the incoming dNTP and not the base – what is the importance of this to the method ?
-This is important because typically when a phosphodiester bond is formed via nucleotide incorporation, the phosphate chain is cleaved and only the alpha phosphate remains in a growing chain
-However, since the fluorescent dye is attached to the gamma phosphate, upon nucleotide incorporation the fluorescent will be shown quickly but then immediately stop once the inorganic phosphate is released with the fluorescence
What is the importance of a ZMW to SMRT
a nanoscale observation chamber is small
enough to observe only a single nucleotide of
DNA being incorporated by DNA polymerase
Explain how single molecule real-time (SMRT) sequencing works
- Each of the four DNA bases is attached to
one of four different fluorescent dyes. - When a nucleotide is incorporated by the
DNA polymerase, the fluorescent tag is
cleaved off and diffuses out of the observation area of the ZMW where its
fluorescence is no longer observable - A detector detects the fluorescent signal
of the nucleotide incorporation, and the
base call is made according to the corresponding fluorescence of the dye - The DNA sequencing is done on a chip/flow cell that contains millions of
ZMWs.
Inside each ZMW, a single active DNA polymerase with a single molecule of
single stranded DNA template is immobilized to the bottom through which light can penetrate and create a visualization chamber that allows monitoring of the activity of the incorporation of nucleotides so that the sequence of the genome can be determined
Explain how read length is a problem for Illumina SBS
Relatively short read lengths due to delays of various methods in nucleotide incorporation which cause low processivity and therefore short read length
* Ultimately short read length can lead to mis-assembly and gaps in genomes with numerous repeated sequences
What is the importance of P5 and P7 in Illumina SBS
- flow cell binding sites that allow the the DNA library fragment to attach to the flow cell surface
What is the main problem of Roche 454 pyrosequencing ?
It is difficult for the system to accurately count the number of bases in
homopolymers (three or more consecutive identical DNA bases) longer than eight or nine bases.
Explain the chemistry behind Illumina Sequencing by Synthesis
- DNA template to be sequenced is randomly sonically fragmented to create the library
- These fragments are then ligated to adaptors and made single-stranded and loaded onto Illumina flow cell/slide where they bind oligonucleotides in distinct nanowells for cluster generation
- Each fragment is amplified on the flow cell (bridge amplification) leaving clusters of clonal single-stranded templates anchored to the flow cell channel
- four labeled dNTPs, primers and DNA polymerase are added to the templates on the flow cell
- once a nucleotide is incorporated, it has a fluorescent blocking group added onto it’s 3’ instead of an OH or H if it was ddNTP and so another nucleotide cannot be added until it is removed
- once the dNTP base pairs with the template, DNA polymerase will incorporate it and leave a fluorescence marker and the identity of the nucleotide that was incorporated can be detected in an imaging system above the flow cell (sequence of the fragment will be known in this way)
- when the phosphodiester bond is made, inorganic phosphate is the leaving group and ATP sulfurylase will use it to form ATP
- After ATP is synthesizes, luciferase is then present which will then create light
- When excited by a laser, fluorescence from each cluster can be detected, which identifies the first base
-wash the entire flow cell after this has been done in each nano well with a nucleotide incorporated and then begin again
Explain the chemistry behind Roche 454 sequencing
- starts off with a DNA template, primer and DNA polymerase
-the genome is sonically fragmented randomly and then THE FRAGMENTS get adaptors with known sequences added onto the ends and then the fragments get attached to flow cells or beads
-The wells on the plates are sized to fit only a single bead
and each 454 picotiter plate contains approximately two
million wells
- the molecules are amplified by PCR to make multiple copies of the template on the same bead
-amplification is necessary because the sequences are fluorescent based and there’s a certain threshold required to detect the fluorescence
- Across the plates, the same exact sequencing reaction is occurring in each well and the correct dNTP that matches the base in the template will undergo base pairing
- Each addition of a new nucleotide is accompanied by the release of pyrophosphate,
which is converted to the emission of light /
chemiluminescence by a reaction including ATP, and luciferase - computational assembly of the sequences of all templates progresses simultaneously
- whenever a nucleotide is incorporated, wherever on the well that produces the light flash would indicate that that specific nucleotide is added into the strand and so that is how you identify what the sequence of the strand is
DNA library
All the genome fragments that will be sequenced in the same reaction
How is template DNA amplified in Roche 454 ? Illumina Sequencing by Synthesis?
- Roche 454 (Pyrosequencing): Template DNA is fragmented, and adaptors are attached to the ends of each fragment. Each fragment is then attached to a single DNA capture bead, and PCR is used to amplify the fragment on the bead to create millions of copies on each bead. After amplification, the beads are loaded onto a PicoTiterPlate for sequencing.
-Illumina Sequencing by Synthesis (SBS): Template DNA is also fragmented, and P5 and P7 adapters are attached to the ends. The fragments are then attached to complementary sequences on the flow cell surface. Bridge amplification, a form of solid-phase PCR, is used to amplify the fragments. In this process, each fragment bends to form a bridge and binds to nearby oligonucleotides, creating clusters of identical DNA copies on the flow cell surface. This amplification forms dense clusters, allowing millions of fragments to be sequenced simultaneously.
Compare the strengths and weaknesses of chain termination (Sanger) sequencing and next-
generation sequencing methods
NEXT GENERATION SEQUENCING
-Advantages = Did not require cloning of DNA fragments in bacteria
* Did not require electrophoretic separation of DNA fragments
* Massively parallel – millions of templates sequenced at the same time
* Relatively inexpensive
Disadvantages = Relatively short reads due to delays in the methods of nucleotide incorporation cause low processivity
and therefore short read length
* Ultimately short read length can lead to mis-assembly and gaps in genomes with numerous repeated sequences
SANGER SEQUENCING
-* Chain-termination method was easy to automate
* Automated sequencers with multiple capillary gels working in parallel can read up to 384 different sequences in one hour
* Note this would require round the clock technical support to prepare sequencing reactions, load sequencers etc
* If this support can be obtained: sequencing a genome can be performed in a relatively short time
- With chain-termination method, at least 5x sequence coverage is needed meaning every nucleotide in the genome should be present in five different reads (MORE ACCURACY)
Disadvantages:
-it was realized that progress in Genomics would be slow if had to rely only on this method and would take months to years for every new species to be sequenced –
* Cost and time also important for genome resequencing projects
Amplification of Immobilized Libraries
- PCR used to produce
sufficient number of
identical DNA copies to be
sequenced - Adaptors provide
annealing sites for primers - Thus the same set of
primers is used to amplify
all DNA fragments in the
library even through the
fragments themselves are
distinct - PCR carried out in the
oil emulsion so that the
products of the PCR are
contained within their
own droplet prior to
deposition into the well
on the plastic strip
What are the differences between a genetic and physical map of a genome?
- Genetic Mapping: based on the use of genetic methods (linkage analysis/pedigree analysis)
- Physical Mapping: Based on molecular biology methods where you directly examine DNA molecules in order to identify the position of sequence features
What is the key difference between a genetic and physical marker?
- Genetic markers (genes and DNA) must be allelic meaning that they have at least two alleles that specify distinct phenotypes
- physical markers (sequence tagged sites/STSs) must be in unique locations in the chromosome genome being physically mapped and the sequence of the STS and the immediate flanking region must be known so that PCR can assay for the presence/absence of the STS on difference DNA fragments
What is an SNP? How are they typed?
- A single nucleotide polymorphism
- A position in genome where some individuals have one nucleotide but then others have a different nucleotide in that same position instead
- Genotypying includes oligonucleotide hybridization (assaying one SNP at a time by using a genome map ad then preparing a DNA target strand from an individual - an oligonucleotide would be engineered to base pair with that SNP site - if base pairing occurs, then that individual has that gene located in that position but if it doesn’t then they don’t have it and would have an SNP at that location instead) and DNA chip (assays more than one SNP at a time using a DNA chip with oligoncucleotides immobilized on the surface of a chip - oligonucleotides would carry both alleles of each SNP - labelled DNA with fluorescent would be applied and positions that hybridization occurs on the chip are determined using a laser)
How were SNP maps engineered for the human genome?
-SNP maps would come after the genetic mapping for humans was done by genes, RFLPs and SSLPs
- This is because it can only be done after the genome is sequenced
- Once you have your first human genome sequenced, the more individuals you sequence afterwards will allow for more information to be available about where SNPs are located because then you can compare each human sequence acquired
What is an STS marker?
- A short DNA (100-500bp) that is easily recognizable/ unique and occurs only once in the chromosome or genome being mapped
Criteria:
1. Sequence of the STS and immediate flanking region must be known so that PCR can assay for the presence/absence of the STS on different DNA fragments
- Must be in unique locations in the chromosome genome being physically mapped
What are the three major types used in physical mapping?
- ESTs: Expressed Sequence Tags
- Short DNA sequence reads obtained by analysis of cDNA clones (complementary DNA to an RNA strand) which is prepared by reverse transcription of mRNA into dsDNA
* Because the mRNA in a cell is derived from protein-coding genes, cDNAs and the ESTs obtained from them represent the genes that were being expressed in the cell from which the mRNA was prepared
* ESTs viewed as a way to get snapshot of the sequences of important genes (gene
discovery)
* EST can be STS if gene is unique and not from a gene family where the genes have
very similar sequences - SSLPs (simple sequence length polymorphisms)
* Genetic marker that can also act as STS
* Those that have been placed on genetic maps provide a direct connection between genetic and physical maps - Random Genomic Sequences
* Obtained by sequencing random pieces of cloned genomic DNA
* Even more helpful if the sequence contains an SNP allowing direct connection
between physical and genetic maps
What is the relationship between an EST and a cDNA
- An EST is a segment of
a sequence from a cDNA clone that corresponds to an mRNA - Because the mRNA in a cell is derived from protein-coding genes, cDNAs and the ESTs obtained from them represent the genes that were being expressed in the cell from which the mRNA was prepared
cDNA library
- collection of cloned complimentary DNA fragments that make up the transcriptome of an organism and they are stored as a library
DNA library
- collection of DNA fragments that compose an organism’s genome and are stored as a library
Outline the approach used to sequence Haemophilus influenza genome
The sequencing of the Haemophilus influenzae genome was the first complete sequencing of a free-living organism, achieved in 1995 using the whole-genome shotgun sequencing approach:
Genome Fragmentation: The entire genome was broken into small, random fragments of DNA.
Cloning: These fragments were cloned into plasmid vectors to create a library, enabling multiple copies of each fragment for sequencing.
Sequencing: The fragments were sequenced from both ends using Sanger sequencing.
Assembly: Computational methods were used to assemble the sequences by aligning overlapping regions, reconstructing the full genome sequence.
What factors complicate the shotgun sequencing approach of eukaryotic genomes compared to
prokaryotic genomes
The fact that eukaryotes have more repeated sequences than prokaryotic genomes, eukaryotes have much larger genomes and would require more and longer sequencing reads
Briefly describe how sequence reads are assembled into contiguous sequence
- A culture of the organism’s genome would be grown
- the DNA would be extracted and then sonically fragmented into various sizes
- the fragmented DNA would be ran on agarose gel to separate them by size
- DNA fragments are picked out that are 1.6 - 2 kb in length
- each fragment is cloned into one pUC18 plasmid to create a library
-paired end sequencing was done using the universal primers on the plasmid - the sequences were then assembled to identify overlaps between the ends of different reads using a computer assembly algorithm and then contiguous sequences were determined (different, non-overlapping parts of the genome with gaps in between them)
What is the mapping reagent in STS mapping?
- The mapping reagent is the collection of DNA fragments spanning the genome being studied
- Mapping reagent is typically clone library and panel of radiation hybrids
Why can an SSLP be used in both genetic and physical mapping
- Because even though they are allelic, PCR assays are used to detect their presence or absence)
- SSLPs can be detected in genomic libraries or physical maps, enabling their use to map the exact physical locations of genes on chromosomes.
Define paired-end reads and indicate their importance to genome sequencing
- Paired end reads are the two resulting sequences provided when both ends of the same plasmid are sequenced
- This is significant because then you’ll have an idea of the sequence that’s between the paired end reads since there won’t be overlap but you’ll be able to know the distance between the paired end reads
- The computer assembly will then take those individual paired end reads with the approximate base pairs between them and do this will all the paired end reads from the other plasmids so that they can be aligned to ultimately get the complete sequence of the fragment
- The process of trying to find paired end reads from a plasmid that links 2 contains together will form a scaffold and then an internal primer is made to complete the internal sequence and link the 2 contains together to form a longer contig
Describe the hierarchal shotgun method for genome sequencing. To what extent do you think this approach is necessary for prokaryotic genomes? Eukaryotic genomes?
- You start with a genomic DNA (come back)
Cycle Sequencing (PCR and Sanger sequencing)
- Cycle sequencing is chain termination sequencing performed in a PCR thermal
cycler - By repeating a cycle iteratively, the amount of each fragment made in the reaction
can be substantially increased. Since each fragment carries fluorescent dyes,
increasing the number of copied fragments also increases the strength of the
fluorescent signal - Cycle sequencing improves the sensitivity of the sequencing reaction, and even very small amounts of starting DNA sample can be used as template – making DNA
sequencing more efficient and cost-effective
= Cycle sequencing requires a heat-stable DNA polymerase.
One key difference between cycle sequencing and PCR is that only one primer is
used in each cycle sequencing reaction so that the amplification of product is
linear, not exponential
- Benefits = you can use less starting material which is cost effective
- more product made which was important because then radiolabels would no longer be necessary
Capillary Electrophoresis and DNA Sequencing
In CE-based DNA analysis, DNA fragments are separated based on their size by running them through a gel matrix. The gel matrix is composed of a polymer such as polyethylene oxide (PEO)
or linear polyacrylamide (LPA)
- Capillary electrophoresis separations are significant because they provide fast separations of limited sample volumes because the capillaries have excellent capabilities to dissipate heat, permitting much higher electric field strengths to be used than slab gel electrophoresis = rapid separation
- capillaries can be easily refilled and changed for efficient and automated injections - you can eject the gel right after it’s used and refill it quickly so that you can run another cycle
- Detection of which. nucleotide is present occurs via fluorescence through a window etched in the capillary
