Amplification Techniques
@Yujeong Oh
Amplification Techniques
PCR
Theories
https://www.sciencedirect.com/topics/neuroscience/polymerase-chain-reaction
PCR stands for Polymerase Chain Reaction. It is a golden-standard technique that is used to amplify a small amount of a target DNA region using thermal cycles. It uses heat-stable DNA polymerase enzyme (Taq polymerase) to make new strands of DNA using existing strands as templates. First step is to initialize DNA polymerase using heat. Then, heat the reaction to denature the double-stranded DNA template by breaking the hydrogen bonds between complementary bases. Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA. Raise the reaction temperature, which enables Taq polymerase to extend the primers, producing new strands of DNA. This denature-anneal-elongation process is repeated 20-40 times to generate multiple copies of target DNA.
Real-time/ quantitative PCR (qPCR) allows the quantification and detection of the specific DNA sequence in real time. It measures the concentration of probes or fluorescent dyes while the synthesis process is taking place.
Protocol
BioRad
Materials needed: BioRad Mastermix (BioRad: #1665009EDU), DNA sample, forward and reverse PCR primers for target gene, nuclease-free water
- Prepare 10 μM working stock of each primer by extracting 5 μl of 100 μM stock and adding 45 μl of distilled water to a total of 50 μl.
- Labelled each PCR tube with name of gBlock
- Add 20 μll PCR Master Mix (BioRad: #1665009EDU) to each tube
- Add 1 μll of 10 μM of the forward primer to each appropriate tube. This is done for all the gBlocks..
- Add 1 μl of 10 μM of the reverse primers to each appropriate tube. This is done for all genes.
- Add 2μl of the gBlock template DNA.
- Add 16 μl of nuclease-free water to reach a total volume to 40 μl
- Set the temperature of each part of the cycle according to the BioRad (35 cycles):
- Cycle 1: 94 °C for 2 min
- Cycle 2 (35 repeats): 94 °C for 1 min
60 °C for 1 min
72 °C for 2 min
- Cycle 3: 72 °C for 10 min
- Hold: 4°C overnight
NEB
Materials needed: NEB Q5 High-Fidelity 2x Master Mix, primers, DNA template, nuclease-free water, PCR machine, pipette
Method:
The following mix is prepared:
- 12.5 μl of NEB Q5 High-Fidelity 2X Master Mix
- 1.25 μl of forward primer (10 μM)
- 1.25 μl of reverse primer (10 μM)
- 2 μl of DNA template
- 8 μl of nuclease-free water
The following settings were inputted to the machine:
- Denaturation: 98 C for 30 sec
- Cycles (35):
- 98 C for 10 seconds
- annealing temperature specific to the gene for 30 seconds
- 72 C for 15 seconds
- Final Extension: 72 C for 2 min
- Hold: 4 C
Optimized NEB PCR Protocol (Taq 2x Master Mix)
- Add the following components in each pcr tube
- Taq 2X Master Mix: 8ul
- Forward Primer (10uM): 0.32ul
- Reverse Primer (10uM): 0.32ul
- Template DNA: 5ul
- Nuclease-free water: 2.36ul (up to 16ul)
- Set the temperature of each part of the cycle according to the BioRad (30 cycles):
- Cycle 1: 94 °C for 2 min
- Cycle 2 (35 repeats): 94 °C for 1 min
60 °C for 1 min (annealing temperature of primer)
72 °C for 2 min
Cycle 3: 72 °C for 10 min
- Hold: 4°C
qPCR
Materials needed: Luna® Universal Probe qPCR Master Mix, PCR primers, Nuclease-free water, SYBR Green, DNA template
Method
- Thaw Luna Universal Probe qPCR Master Mix and other reaction components at room temperature, then place on ice. After thawing completely, briefly mix each component by inversion, pipetting or gentle vortexing.
- Determine the total volume for the appropriate number of reactions, plus 10% overage and prepare assay mix of all components except DNA template accordingly. Mix thoroughly but gently by pipetting or vortexing. Collect liquid to the bottom of the tube by brief centrifugation.
- Aliquot assay mix into qPCR tubes or plate. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles.
- Add DNA templates to qPCR tubes or plate. Seal tubes with flat, optically transparent caps; seal plates with optically transparent film. Care should be taken to properly seal plate edges and corners to prevent artifacts caused by evaporation.
- Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).
- Program real-time instrument with indicated thermocycling protocol (see table below). Ensure a plate read is included at the end of the extension step.
Use the SYBR® or SYBR/FAM scan mode setting on the real-time instrument.
RT-PCR
Materials needed: OneTaq One-Step RT-PCR kit, RNA sample, forward and reverse PCR primers for target gene, nuclease-free water
Method (provided by NEB)
- Thaw system components and mix by inverting several times.
- Mix the following components, except RNA, in sterile RNase-free microfuge tubes.OneTaq One-Step Reaction Mix (2X) 25 μlOneTaq One-Step Enzyme Mix (25X) 2 μlGene-specific Forward Primer (10 μM) 2 μlGene-specific Reverse Primer (10 μM) 2 μlNuclease-free H2O 19–x μlTotal RNA (up to 1 μg) x μl------------------------------------------------------------------------------------------ Total volume 50 μl
- Add RNA template last, and start reactions immediately, as follows:
Primer Design
1.NCBI provides Primer-BLAST for automatically designing primers based on a query sequence.
To start designing primers, go to the BLAST homepage and scroll down to the Primer-BLAST option under Specialized BLAST.
2. Enter your target sequence either by cut-and-paste or, if it’s listed in NCBI’s databases, as an accession number.
3. The target sequence is normally found earlier on by searching the particular gene sequence and downloading the fasta file or copying directly the sequence.
4. The ‘Descriptions’ section of the report now includes a download menu that provides direct access to FASTA data, either of the complete subject sequence or only the aligned portion, as well as to other formats including GenBank flat files, hit tables and XML. Checkboxes to the left of each subject sequence allow users to download an arbitrary subset of the matching sequences. Clicking the title of a subject now links to the alignment display, which also contains similar download functions for that alignment along with controls to navigate between alignments and back to the ‘Descriptions’ table.
5. In the "search for primers dialogue box", gene sequences from the fasta file was pasted and blasted against all genomes. The length of the primers sequence was unspecified neither was the genomes of the organism specified.
6. The primers were designed according to the preset and optimum conditions for Tm.
7. The selection of the right primers for our experiments were done accordingly to the following parameters : GC content = 50% , self 3' complimentarity < 3.00 and self complimentarity < 7.00
Preferred primers had the G-C clamp
8. The complementarity tests were performed using tools from Oligocalc tools.
LAMP
Theories
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC102748/
LAMP stands for Loop-mediated AMPlification and it is a technique that amplifies DNA at a constant temperature lower than for PCR. It accomplishes this by having a different kind of DNA Polymerase, a strand-displacing polymerase, that is capable of separating the strands at a lower temperature and elongating the primers. However, LAMP requires a bit more complex set of primers, because the goal is to form terminal loops on the synthesized DNA. The use of 4 primers that specifically recognize 6 distinct regions on the target gene increases the specificity. When designing the primers, one has to make sure two of them match an exact sequence on the DNA, so that when they are extended, they can form a loop between the primer and the inverse sequence on the new DNA. This process is repeated until both ends have the loops. The new DNA now has 6 possible starting sequences for DNA replication versus 2 in PCR. The loop structure allows us to make complex hierarchical structures capable of amplifying these starting sites, leading to even faster DNA replication.
Protocol
LAMP
Materials needed: isothermal master mix (optigene), nuclease-free water, LAMP primers, Thermocycler, DNA sample
Method (optimized by iGEM NYUAD 2018):
- Prepare the primer mix in an eppendorf tube. Each primer mix consists of:
- 4μl of FIP (100μM)
- 4μl of BIP (100μM)
- 1μl of F3 (100μM)
- 1μl of B3 (100μM)
- 45μl of dH2O in the eppendorf tube.
- Add 15μl of isothermal master mix to each tube.
- Add 5μl of the primer mix to each tube.
- Add 5μl of the dilution sample to each tube.
- Incubate at 65C for 20 minutes.
- Prepare the primer mix in an eppendorf tube. Each primer mix consists of:
Primer Design
A) Select target sequence:
1. Select a highly conserved region of target gene.
2. Target region should be 200 - 2000 bp long.
3. Save the target sequence file in FASTA or text (.txt) format.
B) LAMP Primer design:
1. Launch LAMP primer design software: http://primerexplorer.jp/lampv5e/index.html
2. Upload the sequence file, set the parameter set as "automatic judgement", and click “Primer Design”.
3. (Optional) On the next screen, click on “Detail Settings”. If necessary, select AT-rich or GC-rich option. The Tm of primers may also be varied, but changing the length of primers is not recommended.n
4. Click “Generate” to display number of primers designed.
5. Click on “Display” to display the list of primers in new window. This page lists 1-100 primer sets.
6. To look at additional primers, change the page number, and it will open a new window with list of primer sets. Each page lists maximum of 100 primers.
C) Selecting the primers:
1. Select the desired primer set by checking the box on the left. Multiple primer sets can be chosen at the same time. Lower dG values are usually preferred. Click “Confirm” to select the primer set.
2. A new window will open to show the details of the selected primer set.
Check the stability of the following regions to confirm that the dG is < -4.0 kcal/mol:
* the 3’ end at the region F2
* the 5’ end at the region F1c
* the 3’end at the region B2
* the 5’ end at the region B1c
In the above example, the 3’ end of Primer B1C (dG = -6.26kcal/mol) has the highest stability. The dG of 5’ end of Primer F1c (-3.92 kcal/mol) is above -4.0, indicating it is unstable. Therefore, this entire primer set should not be used. Another primer set should be selected from the list.
3. Once primer sets with required stability are found, check whether F3 and B3 of the set fits into the guide RNA range.
Troubleshooting: If no primers are designed or too few primer pairs are generated, try changing parameters:
1. Use a broader range of Tm for primers, but make sure to keep the Tm of F1c/B1c about 3-4 degrees higher than that of F2/B2 and F3/B3 primers.
2. Change the minimum length of F2/B2 and F3/B3 primers to 15 rather than 18.
3. Change the GC rate to 20 or 25 rather than 40.
RPA
Theories
https://www.sciencedirect.com/science/article/pii/S0165993617302583
RPA stands for Recombinase Polymerase Amplification. It is a technique that amplifies a small amount of DNA at a fast rate and lower constant temperature compared to other techniques such as PCR. It uses a special set of enzymes called Recombinases to insert the primers into homologous or similar sequences on the DNA, which helps start the separation of the strands. A special strand-displacing DNA polymerase then makes sure the primer is extended along the DNA to be amplified requiring no higher temperatures than 37℃. Because the temperature is so low, the separated strands of the DNA can easily rehybridize or re-bind, which will interfere with the DNA replication process. To make sure this does not happen, a family of proteins called single-strand-binding proteins, or SSB proteins, bind to the separated strands to prevent them from reforming hydrogen bonds. As the DNA polymerases extend the primers on both strands, the original DNA strands fully separate, eventually producing 2 new molecules of the same DNA. The steps are repeated for each new DNA molecule and the exponential amplification of just a small amount of starting DNA is achieved.
Protocol
RPA
Materials needed: TwistDx Basic Kit, nuclease-free water, RPA primers, Thermocycler, DNA sample
Method (optimized by iGEM NYUAD 2019):
- Prepare the reaction mix in an eppendorf tube:
- Add the following reagents to the reaction mix:
- Rehydration Buffer - 119µL
- dH2O - 32.8µL
3. Pipette up and down to ensure proper mixing.
4. Add the reaction mix in each tube to four freeze-dried reaction tube (provided by TwistDX Basic Kit).
5. Add 16 μl of the reaction mix to 9 eppendorf tubes.
6. Add 5µL of template from the serial dilutions (from the highest concentration to the lowest concentration) to the corresponding tubes. Leave one as a negative control.
7. Add 1µL of 280mM magnesium acetate to each eppendorf tube and vortex to start the reaction.
8. Incubate the reaction at 38 °C for 20 min using the Eppendorf Thermocycler.
RT-RPA
Reagents/materials needed: TwistDx Basic Kit, nuclease-free water, RPA primers, M-MulV Reverse Transcriptase, RNase inhibitor, RNA sample
Method (protocol provided by Lucia et al. (2020)):
- Mix the following components:
- Primer A (10µM) - 2.4µL
- Primer B (10µM) - 2.4µL
- Rehydration Buffer - 29.5µL
- dH2O - 4.7µL
- Pipette up and down to ensure proper mixing.
- Add the reaction mix to a freeze-dried reaction tube (provided by TwistDX Basic Kit).
- Add the following components to the mix:
- 280mM Magnesium acetate - 2.5µL
- M-MulV reverse transcriptase - 2.5µL
- (Murine) RNase inhibitor - 1µL
- Template RNA - 5µL
------------------------------------------------------------------------------------------ Total volume 50 μl
- Incubate 30 minutes at 42°C
- Mix the following components:
Reaction Conditions
Recommended reaction temperature is between 37C and 42C.
Recommended pH is 7.9
Primer Design
- Genes were located in .fasta format on NCBI and sequenced were created in Benchling
2. For RPAprimers generated from Primer BLAST, the following values were used in settings according to TwistDx recommendations:
Parameter Product Size Primer Melting Temperature Primer Size Range Primer Size Optimal Primer GC Content Value(s) 50-100 30-36 32 30%-70% 3. In addition, a Python package called primedRPA (detailed in the paper titled PrimedRPA: primer design for recombinase polymerase amplification assays, available at https://www.ncbi.nlm.nih.gov/pubmed/30101342) was used with the following parameters:
Please define the reference name for this PrimedRPA run: >run_name Please indicate if you would like to use a previously generated Alignment File: [NO or File path] >NO Please indicate if you would like to use the previously generated Binding Sites: [NO or File path] >NO Please enter the path, from your current working directory, to the input fasta file: >path/to/input_sequence.fasta Please classify the contents of the input fasta file as one of the following options: [SS, MS, AMS]. Whereby: SS = Single sequence MS = Multiple unaligned sequences MAS = Multiple aligned sequences >SS If multiple sequences are present in the input fasta file (Classification of MS or MAS), please indicate below the percentage identity required for the primers and probes target binding sites: >99 Please indicate if a primer identity anchor is required. [NO or length of anchor] >NO Desired primer length (This can be a range: 28-32 or fixed value: 32): >32 Please state if you require a probe to be designed and if so what type [NO,EXO,NFO] >NO Desired probe length (This can be a range: 45-50 or fixed value: 50): >10 Below please define your max amplicon length. >300 Below please state the repeat nucleotide cut-off in bp (e.g. 5bp will exclude sequences containing GGGGG). >5 Below please insert the minimum percentage GC content for primer/probe: >30 Below please insert the maximum percentage GC content for primer/probe: >70 Below please indicate the percentage match tolerance for primer-probe dimerisation and secondary structure formation: >60 Please enter [No or Path to Background file] below to identify if you want to perform a background DNA binding check: >NO Below please insert the percentage background cross reactivity threshold: >65 Below please indicate if you would like to implement a Background Hard Fail Filter [NO,YES]: >NO Please define the maximum number of sets you would like to identify: >5 Please define the number of threads available: >1 Blastn Cross Reactivity Search Settings [Basic or Advanced or Fast] >Advanced Blastn Evalue >1000
4. The primers generated were annoated in Benchling and optimal primers (preferably 1 pair from primer BLAST and 1 from primedRPA) were selected based on location, GC content, fitness with PCR amplicon, and quality of available gRNAs within the amplified region.