PCR - DNA amplification and manipulation

What is PCR?

A DNA replication-dependent means of amplifying relatively short, specific pieces of DNA

What are the advantages of PCR?

• Sensitive, this means tiny start amounts of DNA can be used to produce large amounts of product

• Specific, PCR only produces copies of the target DNA

• Fast, usually between 2-3hours, but new machines can make this as short as 20 minutes

What are the disadvantages of PCR?

• The sensitivity of the process leads to problems with contamination → any other DNA other than the target could also be replicated (if the primers are also specific to this DNA)

• Fickle - trying a range of condition in the hopes that one matches - this is empirical optimisation → not entirely random but is reliant on luck

Describe the process of PCR

• Start with template strand containing the area of DNA you want to amplify (this is the target DNA), a pair of primers, dNTPs, thermostable DNA polymerase, buffers and salts, optional BSA, DMSO and formamide

• Denaturation: heat to 95C, H bonds break between strands, produces ssDNA

• Annealing: reduce to 50-68C, primers anneal to complementary regions flanking the target DNA

• Elongation: thermostable DNA polymerase will move in 5’ → 3’ direction and add free dNTPs

• Repeat around 20-30 times, leads to an exponential increase in the product amount

What is the target DNA?

A small region in the total template DNA that you want to amplify

What are primers?

Short made to order oligonucleotides, complementary to regions of DNA flanking the target DNA - provides specificity of the region of amplification

Why is a thermostable DNA polymerase used in PCR?

This can work at higher temperatures used in all parts of the cycle and can work quickly

What/why are buffers and salts used in PCR?

• KCl and MgCl2

• These maintain the correct pH for the reaction and enzymes

Why are BSA, DMSO and form amide used in PCR?

BSA - some enzymes need for activity

DMSO + formamide alter H bonding abilities of DNA, reducing the temperature needed for denaturation

Describe Taq polymerase and the pros and cons of

• Error prone - no proofreading domain - this is fine if you are only trying to detect if DNA is there or not

• Can be used to generate mutations for forwards genetics

• Not good if you need the exact sequence of DNA for cloning or sequencing as there are random mutations

• Leaves a 3’ A overhang, this can be useful for sub cloning reactions

What is a sub cloning reaction?

These transfer a DNA segment from one plasmid to another

Describe Pfu and Two, and pros and cons

• Accurate as they are able to proofread and are thermostable

• Do not produce overhangs, so blunt end cloning is required

• `lower efficiency than those producing a 3’ A overhang - they are a lot slower, this is due to the requirement of proofreading

• PFU 6X more accurate than Taq

Describe Expand HiFi and 20kbplus

Optimised to do longer range PCR

Describe Fusion

Uses a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain, this increases fidelity and speed

What should be considered when selecting a thermostable DNA polymerase?

• Have varied proofreading abilities and speed, some are able to do longer PCR

• All have different properties, and are often much more expensive than the original ones

What are the guidelines for primer design?

• Two pieces of designed DNA that provide specificty of where in the DNA is amplified

• Design a complementary seqeunce to DNA flanking the region to be amplified

• Must be fairly long (18-30bp) - this ensures specificity must only bind a single site within the template DNA

• Must have a G+C content between 40-60%

• There must be a G or C at the 3’ end, the GC clamp produced stabilises primer binding (3H bonds)→ must also be complementary

• No sequences should be able to result in secondary structures such as hairpins that base pair with themselves (so there cant be any inverted repeats)

• There should be no significant complementarity between primers - this would result in them hydrogen bonding to eachother

• Long sequences of the same nucleotide should be avoided

• Mismatches should be at/near the 5’end, extension end needs to be complementary

• Should aim for an equal Tm of each primer - both are stable at the same temperatures. This ensures that the temperatures for annealing are the same

What is the TM of primers?

Temp at which the primers become single stranded

What is the equation for TM?

TM = 2(A+T) + 4(G+C)

What is the general equation for annealing temperature

TM - 5C

What happens to the TM of primers at varied salt concentrations?

• At different salt concentrations, annealing temperatures of primers alter → altering Mg/NaCl will alter the annealing temperature

• Short primers have higher TM at low salt concentrations

How should a primer be written?

5’ to 3’

How can a primer be designed when we have no genome sequence?

• Can use the protein sequence to guess mRNA

• Can compare the DNA sequence of known genomes

• Can best guess primers with some position having all possible combinations of bases, can use inosine in the primer (inosine is a product of the breakdown of adenosine)

• Degenerate primers: mixtures of different DNA oligonucelotide sequences, used to amplify unknown/highly variable target DNA sequences

What are degenerate primers? Why are they needed?

• Mixtures of different DNA oligonucelotide sequences, used to amplify unknown/highly variable target DNA sequences

• For some amino acids, there is only one codon, but for others there is not - so you have to design a primer mixture with all of the different combinations of the possible DNA code

• Can use artificial bases (like inosine) that base pair to more than one, reducing the numbers of primers needed

• Issues as the probability of binding elsewhere in the genome is quite high

What are RE sites?

Restriction sites can be used to allow RE’s to cut the end and allow cloning into a plasmid/other pice of DNA

Why are PCR primers be designed with RE sites?

May also want to amplify and use DNA for cloning so we can do something with the DNA

How can RE sites be added in via PCR?

Restriction sites can be added to a primer sequence (at the 5’ end) to aid subsequent cloning → Non-complementary sequences must be added towards the 5’ end, as the 3’ binding must be complementary

Cannot add right at the end of the primer as they cut in a site, so you add 2bases at the before the RE site to ensure restriction enzymes cut in the right site → this is becuase restriction endonuclease cut in a seqeunce, not at the end → this is known as linker DNA → 5’ to 3’ - 2 random base pairs, restriction site in primer, complementary DNA

Why can temperature be increased after a few rounds of PCR using RE sites in primers?

Subsequent cycles may benefit from an increased Tm, as DNA is more complementary than the original sample DNA - this is because primers are becoming incorporated into the product which serves as a template in the next round

• The DNA produced in later cycles will be more complementary than those produced before, so increasing the melting termperature will increase specificity as it prevents the inital complementary seqeunce from being replicated again

• In later cycles, whole primer length will be binding, rather than only the complementary part like in the beginning

What computational softwares can be used for primer design?

• Primer BLAST

• Thermofisher multiple primer analyser

• Integrated DNA technologies (IDT) OligoAnalzer

• Sigma OligoEvaluator

• Benchling

• Can all design primers from a given sequence

Describe the use of hot start PCR

• Based on the fact that proofreading can start attacking primers before the reaction has properly started

• Technique used to inactivate DNA polymerase at room temperature → avoids non-specific DNA amplification during the inital set up of the reaction, the enzyme is only active after the first high-temperature denaturation step

How is DNA polymerase isolated/immobilised in hot start PCR?

Use of antibodies (AB is not thermostable, so at higher temperatues will denature and DNA pol can be active) or wax (will melt when it is at the right temperature)

Describe the use of nested PCR

Overcomes problems with primers binding non-specifically to the template - this can be an issue when using genomic DNA as a starting template, as there will be many regions that are complementary

Describe the advantages and disadvantages of nested PCR

Advantages

• Dramatically increases sensitivity and specificity

Disadvantages

• Contamination is a bigger problem becuase of the increased sensitivity

Describe the process of nested PCR

• Uses 2 PCR reactions with 2 pairs of primers

• 1st PCR produces a smaller template containing the target region

• 2nd PCR uses a 2nd pair of primers that amplify the target region → these wont bind to other regions that had the first pair bind

• Increases specificity by using a product of the 1t round of pcr for a second round of PCR

Describe multiplexing

• Multiple reactions in one tube - can test for multiple templates (DNA strands) or multiple targets on the same template

Describe the diagnostic use of multiplexing

• Used to run multiple PCR reactions in the same tube

• If a specific DNA is always present, this can be used to test if the reaction has always been run/has worked

• Diagnostic: negative may mean something, so need to ensure that it is negative because something isnt there, not because the reaction hasn't run

Describe reverse transcription PCR and its process

• Powerful way of making lots of cDNA complementary to the original RNA

• Use reverse transcriptase (viral origin), (RNA dependent DNA polymerase) and use a poly-T primer (binds to the A tail of eukaryotic mRNA) to produce cDNA from the mRNA

• Can then use random primers to amplify the cDNA via PCR. May need to use random primers for the RT process if in bacteria, as they do not contain the polyA tail

• Could also use gene specific primers, allowing you to determine if a specific RNA is present in the sample and also create many copies of cDNA that can be used as probes to determine the mRNA present in the sample/system

• Can all be done in a single tube → just need the right components - RT is dependent on Mn2+, then move to PCR cycle for the rest

How can intron spanning primers be used in RT?

• Can specifically amplify cDNA (made from RNA by reverse transcription) and avoid amplifying gDNA - you can produce DNA that does not contain introns, allowing you to study the functional elements of the gene

• If you design a primer that goes over an exon boundary, it will only bind if you are working with cDNA (as has been made from mRNA that has been sppliced), whereas genomic DNA also contains introns - so will not bind as it is not complementary → so can determine if you are amplifying DNA/RNA, as RNA is spliced

How can we measure how much DNA we started with in a PCR process?

• taking measurements live in the machine during each cycle allows you to measure how much DNA is actually there

• You can’t use the end point measurement - this is because all PCR reactions will reach a plateau (this is because reactant quantities begin to decrease and are now the limiting reagents)

Compare the rate of exponential growth of DNA for small samples vs larger samples of DNA

• Larger samples of DNA will start to exponentially increase before lower amounts, this is because there is more substrate available for the PCR enzymes

What is quantitative PCR (q-PCR), what does it use?

• A method of real-time measuring PCR, allows measurement of initial amounts of DNA

• Uses fluorescent probes (such as Taqman) or SYBR-green incorporation (this intercalates into dsDNA), measures the fluorescence in each cycle
  → increased fluorescence means more DNA

Describe how fluorescent dye-based real time PCR works

Initially, the fluorescent dye binds the dsDNA

Denaturation causes dsDNA to separate

Primers anneal and extension occurs. As extension occurs, more fluorescent dyes are incorporated

• The dye only fluoresces when it is bound to DNA - so more fluorescence means more dye bound to DNA, so more DNA → can use to interpret the concentration of DNA

Describe how fluorescent probe-based real time PCR works

• Probes are attached to the 3’ end of primers

• when DNA polymerase extends the primer, it cleaves off the probe, releasing a fluorescent reporter (a fluorophore). This is causes it to fluoresce

• Binds specific pieces of DNA - can specifically measure the concentration of different pieces of DNA

• Can be used to simeltaneously measure the fluoresence of multiple bits of DNA using different probes and different colours

How can we calculate how much DNA was in an original sample?

Use a standard curve to back calculate how much DNA was in the inital sample

What is the cycle threshold?

The cycle number at which a sample's reaction crosses a fluorescence threshold, indicating the detection of the target nucleic acid

Is inversely proportional to the amount of template in the sample

How is RT-Q-PCR used?

• Can use RNA present (being used to transcribe proteins) to produce cDNA and then amplify by qPCR and detect amount of RNA present

• Used in SARS-CoV-2 diagnosis - when reach over a specific viral load, can diagnose

• Controls: need to link back to a standard to make conclusions

How can PCR be used to do site directed mutagenesis?

Primers have a mismatch, extend and do PCR - making use of PCR produced DNA is not methylated, can then digest the methylated DNA template so you inly have the mutated version

How can forensics use PCR for low copy DNA

• Using regions of the DNA that do vs dont vary

• Only need a small amount of DNA that can be amplified

• Looking at VTRs and amplifying them, can get a pattern of sizes for each person

• Amplified fragment length polymorphism (AFLP) is a DNA fingerprinting technique that analyzes DNA fragments to identify genetic variation between organisms

• You can use PCR to amplify tiny amounts of DNA to the levels required for RFLP analysis

What is inverse PCR used for?

• Aims to amplify unknown regions either side of a known sequence

• Primers are designed outwads from a region of interest - this would not give a product on a linear template

• Must circularise template prior to PCR

• The process is useful in the analysis of DNA regions flanking the gene of interest - can be used to find

How is LAMP an alternative to PCR?

Doesnt require cycling of temperatures

Describe loop mediated amplification and how it works

• Different way of generating ssDNA → pCR does this by heating to a high temperature to break H boinds

• ssDNA is produced by denaturation, addition of a primer that is complementary midway down but not at the 5’ end

• Non complementary creates some signle stranded section

• Start to get regions of complementarity, producing regions where secondary structures form (loops) - the loops that form are ss

• Using multiple complementary regions to produce ssDNA where primers can bind and can be amplified from

• Creating ssDNA by creating DNA structures that are single stranded - rather than using heat

What are the advantages of LAMP over PCR?

• Cheaper and quicker than PCR

• Doesnt require complex machines - doesnt require temperatures

• Conducted in any lab with basic facilites

• Sensitive and reliable → gives the same sensitivity as PCR

• Allows tests in a field → a lot of development for plant pathogens and fungi - somewhat like a LFT