Module 3 Sections 1-5 Flashcards
Section 1: DNA Structure and Function Section 2: RNA Structure and Function Section 3: Quantification of Nucleic Acids Section 4: Amplification of DNA by PCR Section 5: Methods for DNA Sequencing
nucleic acids
composed of chains of nucleotides
basic building blocks for DNA and RNA
Self-assemble into their 3D structure by weak forces and how atoms are arranged in space
3D helical structure of DNA is a result of base pairing and is the most energetically favourable conformation
oligonucleotide
a short nucleic acid containing 50 or fewer nucleotides
polynucleotide
longer nucleic acid
3 components of nucleotides
- A heterocyclic (cyclic compound with 1 or more ring structures that contain atoms of at least 2 different elements) base
- A 5-carbon sugar called pentose
- A phosphate group
chargaff’s rule
A + G = T + C
In DNA, there is always an equal percentage of purines and pyrimidines. This means for all DNA,
of A = # of T
of G = # of C
hydrogen bonding in DNA
2 strands of DNA molecules are held together via base pairing between the nitrogenous bases (hydrogen bonds)
G and C have
3 hydrogen bonds
A and T have
2 hydrogen bonds
DNA double helix
2 strands of DNA intertwine to form a right-handed double helix
Backbone of each strand is composed of alternating sugar and phosphate residues (sugar-phosphate backbone) and is negatively charged
Nitrogenous bases are positioned towards the center of the helix, letting them hydrogen bond with bases on the opposing strand
Has directionality each strand opposing each other (5 to 3 or 3 to 5)
why antiparallel
More energetically favourable than parallel because of the geometry of the component bases
Based on the linkages formed by carbons at the phosphate and OH groups on the pentose sugar
phosphodiester bonds
Link the nucleotide units in nucleic acids
5’-phosphate group of 1 nucleotide linked to the 3’-hydroxyl group of the next nucleotide
major groove of DNA purpose
Nucleotide sequence is primarily read by DNA binding proteins in the major grooves fund on the outside of the DNA strand for more accessibility
2 ways to stabilize the duplex
- hydrophobic stacking
- base pairing
hydrophobic stacking
Chemical properties of purines and pyrimidines
Bases are hydrophobic and are insoluble in water at near-neutral pH of the cell
Thus, bases align so that 2 or more are positioned with the planes of their rings in parallel like a stack of coins (looking down a barrel)
Stabilizes the helix by minimizing contact of the hydrophobic bases with water
base pairing
An extensive network of weak bonds within the double-stranded DNA structure that occurs between base pairs
functions of DNA
- Long-term storage of genetic information
- Acting as a template for DNA replication
- Coding for proteins
what does functions of DNA depend on
Highly dependent on its structure
Once disrupted, it can no longer carry out these critical functions
what makes DNA a good long-term storage of genetic information
- strand complementarity
- replication fidelity
strand complementarity
Hydrogen bonding
Most significant property of DNA that makes it a good information carrier
Specific base pairing within dsDNA allows exact copies to be made, allowing replication of genetic replication
replication fidelity
Structure of double helix can allow for the strands to be separated, and the original is used to synthesize a complementary strand
internal forces on DNA stability
- hydrophobic interactions
- van der waals interactions
- hydrogen bonding between paired bases
- ionic interactions
hydrophobic interactions
Stabilizes base pairing
Bases are hydrophobic and face the interior
Sugar-phosphate backbone is hydrophilic and faces the exterior, interacting with water
van der waals interactions
stacked bases interact through ring structures
hydrogen bonding between paired bases
GC is more stable than AT
ionic interactions
Negative charge of backbone phosphates are neutralized by interactions with cations
Na+ and Mg++ commonly interact with the backbone to neutralize the electrostatic repulsion between strands
external forces on DNA stability
- temperature
above melting temp makes DNA unwind to single strand, destablizing it - salt
increase in salt conc = increase in duplex stability
sodium ions interact with the negatively charged DNA backbone and stabilizes it
- proteins
DNA binding proteins are involved in the compaction of genomes and contribute to both the global and local structure of DNA - organic solvents
Carbon-based
Destabilize DNA helix by disrupting hydrogen bonds and solvating bases
electrostatic interactions using the sugar-phosphate backbone
Due to the negative charge of the sugar-phosphate backbones of both nucleic acids
Backbone of both DNA and RNA is hydrophilic, so the hydroxyl groups of the sugar residues form hydrogen bonds with water
phosphate groups at pH 7
have pKA near 2
completely ionized
negatively charged (neutralized by ionic interactions with positive charges on proteins, metal ions, or short linear organic molecules called polyamines)
polyamines
2 or more amine groups
types of coding RNA
mRNA
mRNA
Transient carriers of genetic information
Transcript copy of a gene that encodes a specific protein
Carries the encoded information from the nucleus to the ribosomes where the protein is produced
Coding sequence of mRNA determines the amino acid sequence of the protein
Different mRNA molecules adopt different 3D structures depending on what is most energetically favourable
non-coding RNA
Transfer RNA (tRNA)
- Present during translation
Ribosomal RNA (rRNA)
- Present during translation
Long non-coding RNAs (lncRNA)
- Can be important regulatory RNAs
Small nuclear RNAs (snRNAs)
- Play a role in gene regulations (splicing “snurps”)
MicroRNAs (miRNAs)
- Limit translation by binding to the 3’-end of target mRNAs
Small interfering RNAs (siRNAs)
- Can inhibit transcription of certain genes and viral DNA
Catalytic RNAs
- Ribozymes
RNA vs DNA similarities
both carriers of genetic information
DNA vs RNA
DNA - long term storage of genetic information
RNA - more transient (less permanent)
DNA - set of biological blueprints
RNA - helps carry out the guidelines found within these blueprints
DNA - double stranded
RNA - single stranded
DNA - not as versatile
RNA - structurally and functionally versatile
pentose sugar
called deoxyribose
In RNA, the deoxyribose sugar is replaced with ribose
Ribose is a 5-carbon sugar with a hydroxyl group at the 2’ carbon. This provides an additional site for hydrogen bonding, stabilizing the 3-dimensional folding of the polynucleotide
RNA base composition
RNA has uracil instead of thymine
non-canonical RNA base pairing
RNA can sometimes have A-A and G-U base pairing (helps stabilize the 3 dimensional folding of RNA, with surfaces capable of binding other molecules)
when is RNA less stable
under alkaline conditions (pH > 7) because of the additional hydroxyl group on the 2’ carbon of the pentose sugar (hydrolyzed rapidly, but DNA is not)
products of action of alkali on RNA
cyclic 2’,3’-monophosphates
cyclic 2’,3’-monophosphates yield the mixture
rapidly hydrolyzed further to yield a mixtuer of nucleoside 2’- and 3’-monophoshates
DNA backbone
serves a purpose in gene expression
DNA is maintained during cell division and during extended periods in nonreplication cells
DNA and RNA in alkaline conditions
DNA: resistant
RNA: degraded
RNA folding
secondary structure, can fold back on itself to form intramolecular base pairings
3D to fold into many different shapes
energetically favourable RNA structures
RNA base stacking hides the hydrophobic bases away from the hydrophilic surroundings
RNA base stacking in tRNA
all the bases are stacked.
base-triple interactions, helix-helix packing allows stable 3D folding
RNA secondary structures
- helical structures
- internal loops
- hairpin loops
helical structures
when the strand folds back onto itself, the paired strands are antiparallel to one another and form a right-handed helix
internal loops
separation of double-stranded DNA (single stranded RNA that folded back on itself) because lack of base pairing
hairpin loops
RNA folds back but there is an unpaired loop of bases at the end of a stem region
nucleotides within loops are arranged to maximize hydrogen bonding and base stacking, enhancing thermodynamic stability
most common type of RNA secondary structure
hairpin loops
stability of RNA structure
influenced by weak interactions - van der waals stabilize structures
metal ions in the stability of RNA structure
bind to specific sites to help shield the negative charge of thebackbone, allowing RNA to tightly pack together
other influences to the stability of RNA structure
- # of GC vs AU base pairs
- # of base pairs in a stem region
- # of base pairs in a hairpin loop (more than 10 or less than 5 bases requires more energy)
- # of unpaired bases - decrease stability
most accurate method of nucleic acid quantification
UV absorption - the light absorbed is directly proportional to the amount of proteinnucleic acid present in the sample
purines vs pyrimidines structure
purines = 2 rings
pyrimidines = 1 ring
ring structure
alternating single and double bonds that create resonance (partial double-bond character)
pyrimidine and purine structure
pyrimidine = planar
purine = nearly planar with a slight pucker
spectrophotometry
measures the amount of light absorbed by a sample
spectrophotometry steps
- Sample containing nucleic acids is placed inside a chamber and is exposed to ultraviolet light at a wavelength of 260 nm
- A photo-detector measures how much light passes through the sample – represented on an absorption spectrum
UV light and nitrogenous bases
they absorb MORE light. the close interaction of stacked bases when bound within a nucleic acid decreases the absorption of UV light relative to that of a solution with the same conc. of free nucleotides
beer’s law
calculating the concentration of nucleic acids in a solution - the darker a solution, the more concentrated it is as it is absorbing more light
beer’s law equation
A260 = e260 * c * l
A260 = the absorbance of UV light as determined by the spectrophotometer
e260 = extinction coefficient
c = concentration
l = path length / distance that light travels through
optical density
the amount of UV light able to pass through a solution
greatest at 260 nm - larger for DNA than for protein
order of UV absorption
dsDNA < ssDNA «_space;free nucleotides
hypochromic effect
Large decrease in light absorption at 260 nm occurring as single strands of DNA anneal to form double-helical DNA
Forces that stabilize the DNA helix like hydrogen bonding and base stacking, limits the amount of resonance that can occur within the aromatic rings of the bases
Decrease in UV light absorbed as ssDNA anneals to form dsDNA
hyperchromicity
Large increase in light absorption at 260 nm that occurs as DNA unwinds/melts
As DNA is denatured, the base pairs are disrupted and the 2 strands separate, forming ssDNA
The resonance within the bases are no longer constrained, so the UV light absorption of a single-stranded DNA is higher than double-stranded at the same concentration
dsDNA viscosity
highly viscious at pH 7 and 25 degrees cels
exposed to extreme pH or above 80 degrees
viscosity decreases, indicating denaturation
dsDNA denaturation
melting and 2 strands break apart - hydrogen bonds and base stacking interactions are disrupted
does DNA absorb more light in single or double stranded form
single-stranded form - the resonance within nitrogenous bases are no longer constrained by the forces that stabilize the DNA double helix
how to determine the melting point of DNA
- track the absorbance of an aqueous solution across a range of temperatures
- the temp at which half the DNA in the sample is denatured (50% dsDNA and 50% ssDNA)
denaturation/melting point uses
- determining base composition
- analyzing structure and function of DNA (interactions holding the strands together)
- estimating the amount of G and C bases
GC base pairs vs AT base pairs in melting points
higher content of GC = higher melting point (3 hydrogen bonds instead of 2)
melting temperature equation
T = 0.41 (%G+C) + 69.3 degrees
under standard conditions and a salt conc. of 0.2 M
renaturation
reconstruction of a nucleic acid (or protein) to its original form after denaturation
reannealing
- 2 strands must not be completely separated (dozen residues must remain intact)
- when pH/temp returns to normal, the separated segments of the double helix spontaneously rewind to form an intact duplex
renaturing completely separated DNA
- Complementary sequences in the 2 strands find each other by random collisions and form a short segment of double helix
- The remaining unpaired bases come together as base pairs and the 2 strands zip themselves together to form the double helix
how are RNA-DNA hybrids made
2 samples of isolated DNA or RNA are heated to cause denaturation and then are mixed. the sample is left to cool, letting the complementary strands to form a duplex whether it is a DNA-DNA, RNA-DNA, or RNA-RNA hybrid
factors that influence DNA hybridization
- DNA concentration
More concentrated sample of DNA has more frequent intermolecular collisions = faster hybridization rate - base pair mismatch
When hybridizing 2 nucleic acid segments that are completely complementary, mismatched nucleotides can interrupt base pairing, stacking interactions, and DNA stability - pH
In a range of 5-9, there is only a minor effect on DNA hybridization
pH < 5 causes the liberation of all bases from the helix
pH > 9 causes the conversion of dsDNA to ssDNA
stringency of DNA hybridization
The extent that hybridization can occur between 2 non-complementary strands of nucleic acid - i.e the degree of complementarity required between 2 strands in order for them to hybridize
high stringency
Hybridization occurs only when the 2 strands are highly compatible
low stringency
Hybridization occurs even in the presence of some base mismatches
increased stringency is used when
Duplex formation is not favoured
High temperatures – close to melting point
Low-salt concentrations
Presence of organic solvents
decreased stringency is used when
Hybrid formation is favoured
Low temperature – below melting point
High-salt concentrations
Absence of organic solvents
what does PCR rely on
DNA polymerases (enzymes able to synthesize DNA strands using a pre-existing DNA template and free deoxyribonucleotides. They add nucleotides (primers) to pre-existing strands)
primers used in PCR
2 synthetic primers are prepared, complementary to sequences on opposite strands of the target DNA at positions defining the ends of the segment to the amplified
extended by a DNA polymerase
simple PCR reaction mixture contains:
DNA sample containing the segment to be amplified
A pair of synthetic oligonucleotide primers
Deoxynucleoside triphosphates (dNTPS)
DNA polymerase
PCR procedure steps
- denaturation
Briefly heated to separate the strands
Enables the components of the reaction to interact with the single-stranded DNA template
- annealing
Cooled so the synthetic primers can anneal to the DNA template
High concentration of primers increases the likelihood that they will anneal to each strand before they can reanneal too one another
- elongation
Temperature is slightly increased for synthesis of a complementary DNA strand
DNA polymerase recognizes the 3’ ends and extends the strands along the targeted segment
- amplification
Steps 1-3 process is repeated for a second time - repeat
Steps 1-3 are repeated 25-30 times, amplifying the DNA segment between the primers until it can be analyzed or cloned
the design of primers
comprised of short sequences of oligonucleotides that are synthesized commerically
parameters for designing a good PCR primer set
- 18-25 nucleotides in length
- 40-60% GC content
- Annealing temperature in the range of 50-60 degrees Celsius
- 1 or 2 GC residues at the 3’ end of the DNA strands
- Minimal secondary structure and base repeating
- Complementary to sequence chosen for amplification
melting temp formula for oligonucleotides
Tm = 2 degrees (A+T) + 4 degrees (G+C)
annealing temp formula
Ta = Tm - 5 degrees
what is gel electrophoresis used for
determine the size of the PCR product
gel electrophoresis
Technique for separating mixtures of large charged molecules like proteins or nucleic acids by causing them to move through a gel matrix when an electric field is applied
gel electrophoresis steps
- The sample of interest is added to a slot at one end of the gel. The gel matrix is composed of agarose (a kelp-derived material that does not disrupt nucleic acid base pairing)
- Voltage is applied to the gel. Since the backbone of nucleic acids are negatively charged, both DNA and RNA will migrate towards the positive end of the gel in the electric field
- Larger molecules tend to move more slowly than smaller ones, so the molecules are separated based on their size – larger molecules are retained closer to the top and smaller molecules migrate towards the bottom of the cell
gel electrophoresis: ethidium bromide
used to visualized the PCR product loaded into each well - fluoresces when exposed to UV light, used to detect nucleic acids in a gel
gel electrophoresis ethidium bromide intercalating agent
planar molecules = inserts itself in between nucleic acid strands in a nonspecific manner
reverse transcriptase PCR
used to amplify segments of RNA like mRNA gene expression products
RT PCR characteristics
RNA is not amplified by PCR, instead it is converted to cDNA, amplified to PCR
can amplify and sequence gene segments without introns
can quantify mRNA levels as a measure of gene expression using quantitative PCR
what does quantitative PCR use
SYBR green
SYBR green
a fluorescent label that fluoresces substantially brighter when bound to dsDNA
SYBR purpose
allows the quantification of dsDNA product inthe reaction after the extension stage of every PCR cycle
measures the amount of PCR product present - the more double stranded products produced with each cycle, the more fluorescence detected
quantitiative PCR steps
- DNA is denatured at 95 degrees in a solution containing SYBR green
- Primer annealing (55-65 degrees)
- Beginning of synthesis
- Extension (60-72 degrees)
- Repeated
cycle threshold
cycle number at which the threshold is first surpassed - should not be reached without target DNA or cDNA
no template control (NTC)
added in quantitative PCR to control the amplification of non-specific DNA products
problems with qPCR
SYBR green may bind to non-desired amplification product and produce a florescent signal
Non-specific products will melt at a different temperature than the desired PCR product, giving a melt curve with sharp decreases at more than one temperature
what is sanger sequencing also known as
dideoxy chain-termination method
sanger method purpose
method for determining the nucleotide sequence of DNA
what does sanger sequencing require
DNA polymerases (to mock the mechanism of DNA synthesis - the 3’-hydroxyl group of the primer reacts with an incoming nucleotide to form a new phosphodiester bond )
labelled primer and dideoxynucleotides (for enzymatic synthesis of a DNA strand complementary to the strand under analysis)
dideoxynucleotides
chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing
why are dideoxynucleotides used in sanger method?
ddNTPS (have H at both 2’ and 3’ positions) interrupt DNA synthesis because they lack the 3’-hydroxyl group needed for the next step
steps of sanger sequencing
- DNA Denaturation
- Apply heat to DNA sample causing the dsDNA to ssDNA, forming template and complementary strands - Primer
- DNA primer is annealed to the template strand, allowing nucleotides to be added later
- Primer is added because DNA polymerase requires a free 3’ hydroxyl group to add free nucleotides
- Primer was radiolabeled in original Sanger sequencing, which allowed the products of the DNA synthesis to be detected by an autoradiogram - Free Nucleotides (dNTPS)
- 4 reaction mixtures are set up and the template strand (with primer) is added to each one along with DNA polymerase and free nucleotides - Modified Nucleotides (ddNTPS)
- Used to terminate the synthesis reaction (no 3’-hydroxyl group, has only –H)
- ddNTPS are added to each reaction mixture
- Only 1 type of ddNTP is added to each reaction mixture, ddATP (green), ddTTP (red), ddCTP (blue), or ddGTP (yellow)
- Added at much lower concentration than dNTPS, allowing for the extension of the synthesized strand with unmodified dNTPS before a ddNTP is added to terminate the extension - Chain Termination
- ddNTPS lack a 3’-OH group required for the formation of a phosphodiester bond between 2 nucleotides
- Causes DNA polymerase to cease extension of DNA - Gel Electrophoresis
- Sample is collected from reaction mixtures
- DNA is separated by size using gel electrophoresis
- Each reaction mixture is added to a separate lane of the gel; the radiolabeled primer is detected by autoradiography
- All possible chain lengths produced are separated by 1 nucleotide
- Shorter fragments run further on the gel than the longer ones
dye-termiantor sanger sequencing vs. sanger sequencing
- All ddNTPS are added to the same reactin (blue, green, yellow and red)
- Products of different size are separated by size using capillary electrophoresis
The fluorescently labeled segments are excited by a laser and the wavelength of the fluorescent emission (red, green, yellow or blue) is detected, one nucleotide at a time
how is dye-termiantor sanger sequencing different from sanger sequencing
- all ddNTPS are added to the same reactin (blue, green, yellow, red)
- Products of different size are separated by size using capillary electrophoresis. The fluorescently labeled segments are excited by a laser and the wavelength of the fluorescent emission (red, green, yellow or blue) is detected, one nucleotide at a time
in vitro PCR amplification
For DNA segments flanked by regions with known sequences, PCR primers can be designed to amplify the region of interest
If the sequence is unknown, synthetic adapters with known sequences can be ligated (enzyme joining 2 nucleic acid fragments) to the ends to serve as primer binding regions, enabling PCR amplification
in vivo DNA replication
Segment is incorporated into a vector by a process called molecular cloning
Once inserted into bacteria, the vector can be replicated as the bacteria proliferate, enabling amplification of the DNA segment of interest
molecular cloning
method to provide large quantities of purified DNA for sequencing
general idea of molecular cloning
Isolation and generation of recombinant DNA molecules that are placed in organisms for replication and study
Separates a specific gene or DNA segment from a larger chromosome and incorporating it into a small molecule of carrier DNA
modified DNA is introduced into a host cell and replicated
steps of molecular cloning
- Obtaining the DNA segment to be cloned
- Enzymes called restriction endonucleases act as precise molecular scissors, recognizing specific sequences in DNA and cleaving genomic DNA into smaller fragments suitable for cloning
- Alternatively, genomic DNA can be sheared randomly into fragments of a desired size
- If the sequence of targeted genomic regions is known, some DNA segments that will be cloned are simply synthesized - Selecting an appropriate carrier molecule of DNA capable of self-replication
- Called cloning vectors and act as carriers of new DNA
- Ex: plasmid vectors and bacterial artificial chromosomes - Joining 2 DNA fragments covalently
- The enzyme DNA ligase links the cloning vector to the DNA fragment to be cloned (inserted)
- Composite DNA molecules of this type, comprising covalently linked segments from 2 or more sources are called recombinant DNA - Moving recombinant DNA from the test tube to a host organism
- The host organism provides the enzymatic machinery for DNA replication
- Bacteria are often used for this purpose - Selecting or identifying host cells that contain recombinant DNA
- Cloning vectors have features that allow the host cells to survive in an environment where cells lacking the vector would die (ex: antibiotic resistance)
- Cells containing the vector are selectable in that environment
cloning vector
DNA molecule known to replicate autonomously (self-replicate) in a host
recombinant DNA
a segment of DNA is placed within a cloning vector, allowing for its replication
engineered plasmid DNA
circular DNA molecule found in bacteria that replicates separately from the bacterial chromosome
when are plasmids useful
when cloning fragments are less than 15,000 bp in length
plasmids purpose
Plasmids have specialized sequences that enable them to use the cell’s resources for their own replication and gene expression to survive
Naturally occurring plasmids have a symbiotic role in the cells (ex: performing new functions for the cell, resistance to antibiotics, etc)
components of plasmid DNA
- Ori (Origin of replication)
- Sequence where replication is initiated by cellular enzymes
- Required to propagate the plasmid - Restriction Sequences
- Several unique restriction sequences are targets for restriction endonucleases
- Providing sites where the plasmid can be cut to insert foreign DNA - Number of Base Pairs
- Facilitates both its entry into cells and the biochemical manipulation of the DNA
- Generated by trimming away many DNA segments from a larger parent plasmid (sequences that the molecular biologist does not need) - Antibiotic Resistance
- Have genes that confer resistance to antibiotics tetracycline and ampicillin
- Allows the selection of cells that contain the intact plasmid or a recombinant version of the plasmid using these antibiotics
stages of molecular cloning experiment
- plasmid generation
- transformation and antibiotic selection
plasmid generation steps
- The pBR322 plasmid DNA is cleaved at the restriction site by a restriction endonuclease called Pstl. The foreign DNA contains the Pstl complementary ends
- Foreign DNA fragments are ligated into the plasmid. Successful integration disrupts the AmpR gene, making the plasmids no longer resistance to ampicillin
transformation and antibiotic selection
- Plasmid DNA is introduced into the bacterial cells by transformation, whereby the cells and plasmids are incubated at 0 degrees in a calcium chloride solution and then heat shocked by raising the temperature to 43 degrees (chemical transformation). Alternatively, the cells can be subjected to a high-voltage pulse to allow the plasmid DNA to enter (electroporation transformation)
- The cells are grown on agar plates with tetracycline to select only for those that have taken up the plasmid. Individual colonies are transferred to matching positions on additional plates. 1 plate has tetracycline and the other has tetracycline and ampicillin
- Cells that grow only on the tetracycline agar plates contain recombinant plasmids with disrupted ampicillin resistance, hence the foreign DNA. Cells with pBR322 without foreign DNA retain ampicillin resistance and grow on both plates mean that cloning was unsuccessful
bacterial artifical chromosomes (BACs)
Have origin of replication, antibiotic resistance, restriction sites and a reporter gene (enables the detection or measurement of gene expression)
BACs have stable origins of replication to support very long segments of cloned DNA
limitations of sanger sequencing
Slow and expensive
Read lengths are only up to 1000-1500 bases
Sequences for a large segment of DNA need to be broken down, analyzed one at a time and compiled together
next-generation sequencing purpose
rapid sequencing of large DNA segments
next gen sequencing steps
- These large DNA segments (could be an entire human genome) are fragmented to smaller segments (300-400 bp) and are sequenced simultaneously
- The sequences for overlapping fragments are then aligned to generate a consensus sequence for the entire DNA segment
what is used in reversible terminator sequencing
modified nucleotides bearing a reversible terminator (RT)
RT sequencing vs sanger sequencing
sanger = labeled with fluorescent tags
RTS = the fluorescent tags are bound to the modified nucleotides using a cleavable linker region
steps of RTS
- library preparation
- cluster generation
- sequencing
- data analysis
RTS library preparation
DNA fragmentation:
- Large DNA segments are cleaved into 300-400 base pair fragments
- Fragmented ends are repaired and a single adenosine nucleotide is added to the 3’ ends of the fragments to prevent them from ligating to each other
adaptor ligation:
- Ligated to the 5’ and 3’ ends of the fragmented DNA segment
- Terminal sequences = essential to the next stage of sequencing, known as Cluster Generation
- Index sequences = allow DNA libraries from different samples to be processed and separately and pooled together in the same run
- Each index is like a unique barcode for DNA fragments from a specific sample. After the run, the sequences from individual samples can be separated from each other based on this indexing barcode
- Primer binding sequences = serve as binding regions for sequencing primers
- Allows for paired sequencing from both ends of the DNA fragment
RTS cluster generation
Involves the amplification of individual sequences from the DNA library to form clusters of clonal (all from the same gene sequence) DNA segments
Takes place in the flow cell, a piece of acrylamide-coated glass that is coated with oligonucleotides
RTS cluster generation steps
- DNA library is added to the flow cell. terminal sequences in the adapters allow single DNA segments to hybridize with the oligonucleotides bound to the surface of the flow cell. a DNA polymerase is used to extend an oligonucleotide that is complementary to the bound DNA molecule
- the original template is washed away, leaving only the newly synthesized strand that is covalently bound to the flow cell
- the adapter sequence at the 3’ end of the bound DNA molecules hybridizes with a nearby oligonucleotide. the bridge is extended and the 2 strands are denatured
- process is repeated, forming a cluster of forward and reverse strands - reverse strands are hydrolyzed and washed away, leaving a cluster of unidirectional clonal strands
RTS sequencing
3 components added to the flow cell:
- 4 fluorescently labeled reversible terminator nucleotides (RT-dATP, RT-dCTP, RT-dGTP, RT-dTTP)
- a sequencing primer that can hybridize with the 3’ adapter region of the bound DNA segment
- DNA polymerase
results of RTS sequencing
graph with many coloured dots - each dot represents a stage of amplification and the colour determines the next nucleotide (e.g, green cluster = next nucleotide is A)
RTS data analysis
aligned to a reference genome once all the reads are generated
“depth” of coverage = # of times that a specific base pair appears in a sequence read
