Bioc2004 Genomics in Diagnostic Microbiology:
Real-time quantitative PCR based assay design
Exercise 1. Primer Design for standard three stage PCR using Primer3plus web interface appropriate for Sybr Green based assay
Exercise 2: Real-time Quantitative Polymerase Chain Reaction (Real-Time qPCR) using an internal probe such as a Taqman assay
Polymerase Chain Reaction (PCR)
The polymerase Chain reaction is an enzyme based DNA amplification process utilizing one of several different thermal polymerases derived from a thermophylic bacterium such as Thermus aqaticus (hence Taq polymerase). However, there are now a range of different polymerases showing thermal stability which are used for this purpose, including some with proof-reading ability confering greater fidelity (accuracy) although these tend to have lower processivity and therefore sensitivity.
The thermal polymerases used in PCR are characterised by their DNA dependent DNA polymerase activity requiring a partially double stranded molecule with a 5' overhang providing a template strand. They thus elogate in a 5' to 3' direction extending the molecule formed usually by annealing a single stranded primer to the template following denaturation of the DNA within the reaction.
The ability to under go multiple rounds of
denaturation, primer annealing and elongation i.e. heating and cooling is an essential characteristic conferred by the enzymes high thermal stability. The primers used in PCR are usually chemically synthesized oligonuleotides of between 18 and 30 bases in length and are specific and complementary to the 3' ends of the forward and reverse strands of the double stranded template sequence to be amplified. Thus the primers are sometimes refered to as the forward and reverse primers. It is critical for the amplification process, that the primer is complementary; but this is particularly true of complementarity at the 3' end and a primer. One or more mis-matches at its 3' terminus and a primer will NOT form an initiation complex that will function in PCR, this is not the case for a primer with 5' mis-matches.
diagramatic representation of three stage cycling of PCR appropriate to a Sybr Green based assay
PCR has become one of the standard molecular biology tools for amplifying segments of DNA and manipulating these for cloning and many other applications. Because of its sensitivity it has become the tool of choice in many diagnostic applications in microbiology. However, the basic PCR technique does not have a quantitative output and cannot be used to inform template copy number. This is because PCR reactions rapidly become rate limited by the conditions within a reaction and the end point therefore does not reflect the initial concentration of template. We therefore use modifications of this technique to provide
measurable output during the exponential non-rate limited phase of the amplification. This can be done in “real-time” and hence the term "real-time PCR" but is better refered to as real-time quantitative PCR (qPCR). Even so, the polyerases used in qPCR are DNA depenent enzymes, PCR will not use RNA as a template. Amplification of specific RNA sequences require that the RNA is first converted to complementary DNA (cDNA) using reservse transcriptase (RT).
The effective and specific amplification of a DNA segment is dependent upon the specificity of the primer sequences used, this means that primer design is a critical step in the process and poor primer design will lead to either non-specific amplification of multiple DNA segments, the wrong segment or failure to amplify anything at all.
The criteria used in the primer design process include
- A specified matched melting temperature (Tm) for both primers
- Lack Self Complementarity, if self complementarity is present this may result in internal intra molecular structure would have an adverse effect on the amplification
- Lack Primer-Primer complementarity (3’ end complementarity should be avoided, this complementarity results in the amplification of primer dimer products in preference to amplicon)
- The primer binding site should be unique within the target molecule
- The combination of primer binding sites should be unique within the population of molecules being used. If both primers have complementarity to a non-target molecule such as another organism they should have opposit orientation to each other or be sufficiently distant as to render them in effective at amplifiying such a target. Primer complemetarity to non-target sequence can be identified by searching a database of sequence entries using BLASTN or some similar program
In practice primer design is done using software. Many different packages are used but one of the most commonly used non-commercial package is called Primer3. Primer3 can be accessed over the internet, it is a very versitile package for designing oligonucleotides for various applications including PCR, real time PCR and sequencing
Notes(click here for pdf version of revision notes)
Exercise 1. Primer design for a Sybr Green real-time qPCR assay
For many applications, including diagnostics the sensitivity of PCR has been further extended and made quantitative by the use of a fluorescent intercalating dye such as Sybr Green. This is important for many applications where not only is sensitivity and specificity important along side a diagnostic threshold but also the amount of target DNA is critical to the clinical response such as in measuring viral load in some assays such as HIV.
Intercalating dyes such as Sybr Green are used in real-time qPCR to quantiatively detect accumulating product usually in a traditional three stage PCR reaction.
Primer Design for HPV-6 PCR using Primer3plus web interface.
More than 100 different HPV types are known and are identified by a number. Some types are more clinically important than others. For example types 6 and 11 cause around 90% of genital warts and are also associated with oral papillomas. There are two vaccines currently in use Cervarix providing protection against two HPV types linked to cervical neoplasias and Gardasil in addition providing protection against a HPV 6 & 11 . Cervarix is the current vaccine of choice adopted by the NHS in England and Wales. The HPV vaccination programme started in September 2008.
- Now for the first exercise we will design primers to amplify a target region of HPV 6 which has been indicated by sequence alignment to provide some specifictiy that virus type. The rational behind this choice we introduced previously, all we need to know for the time being is that it is located within the middle of the genome as represented below, covering a region before the end of E5a and after the start of E5b.
From the NCBI HPV Type 6 Genome entry (there is a link to the NCBI genome viewer here)
- Open the text file HPV6seq.txt by clicking on the hyperlink. Then return back to this page without closing the browser tab/window.
- Open the primer3plus web interface. Note the interface has a series of Tabs (Main, General Settings, Advanced Settings .....), ignore these for the time being and we will use the default values. Copy the sequence and paste it into the text box in the primer3plus interface. Notice that on the upper left there is a drop down box, the default is "Detection". You do not need to change this, but take a look at the options. The page should look something like that below.
- Now run primer3 by clicking on "Pick primers ".
- The page will show the sequence with the first pair of primers highlighted i.e. the forward primer and complement of the reverse primer. The "best" primers and their statistics are given at the top of the page. Up to 5 primers may be listed with warnings/comments below this. If no suitable primer pairs are found none are listed.
- The results page is divided into sections relating to each pair of primers produced by the software. Take a look at the sequences produced, all of the primers are actually very similar. For the top pair both primers have runs of contiguous nucleotides and in particular the left primer contains a run of ts' at its 3' end. This is undesirable. Can you guess why this might be so? Click the "back" button in the interface on the upper left.
- Now study the sequence you will see in the center of the first line of sequence a run of four ts highlight these and click on the button underneath the text box marked "<>". This inserts angled braces around the highlighted region these can also be typed in at the appropriate places in the sequence. The angled braces identifies within the sequence regions to be excluded by the software for the purpose of primer design. Identify any other similar regions of 4 or more similar contiguous sequences. The interface should now look something like that below.
- We are also going to now adjust some of the parameters the software uses such as the product size range. Click on the "General Settings" tab. The text box at the top labeled "Product Size Ranges", replace this content with "100-200".
- Now click "Pick Primers"
- You will notice that the "Any" value has fallen from 6.0 to 3.0 for the left primer and that the primers contain fewer contiguous runs of the same nucleotide and in particular are more heterogenious at their 3 prime ends. One of the problems here is there is very little scope for shifting the primers within the sequence because it is so short to do so and thus restricts our options.
- The next problem is how to address the specificity of the primers!. We have picked a region that is specific to HPV 6, however that doesn't mean that there is not local homology corresponding to the location of the primers and therefore the poossibility of mis-priming. The best way to do this is to identify if the primers can prime other sequences by asking the following:
- Do they have high similarity, especially 3' complementarity with other sequences.
- Do both primers have similarity to the same segment of DNA and on opposit strands in close proximity such that they could amplify a segment of DNA
- If either of these are true different primers need to be chosen from the list or designed.
- This can be tested by carrying out a sequence similarity search essentially a BLAST search. Now go back to the Primer3 results and click on "Send to Primer3Manager" button on the left of the page in the section for "pair 1"
- A new window will open showing the primers something like that below. Choose the first primer Click on the hyperlink "BLAST" on the right hand side.
- This opens a new window containing the BLAST interface at the NCBI.
- The default database is "other" this is essentially all sequence contained within GenBank.
- We will do a Megablast query so click on the radio button towards the bottom of the page "Highly similar sequences (megablast)". This search is optimised for short sequences such as primers and generally works better for this type of comparison.
- Next to the BLAST button check the box "show results in new window"
- Only NOW click the BLAST button
- The results page will eventually open, this page is divided into three parts:
- A graphical representation of the top 50 or so hits. Because there are so many HPV sequences in the database this is less useful in this case but where less redundancy in the database occurs this can be a very useful summary. Complementarity to the 3' end of the primer can be most easily seen from graphic. The horizontal bars represent the hits aligned against the length of the primer (red bar with black numbering) numbered at the top of the graphic. So a gap toward the right hand end indicates a mis-match at the 3' end of the aligned sequences
- The next section is a list of matching entries and associated scores for the individual alignments. Complemetarity oever the entire length is given by the column headed "Query coverage" not "Max ident" which refers to the % identity over the complemetary part of the alignment)
- The lower section has the actual alignments. Note each alignment states values for the score, expect, identities, gaps and STRAND. This latter information tells you if the match is against the sense or complementary (minus) strand in the database.
- Note which hits/sequences have 100% complementarity. . You may need to scroll down to the alignments and check these in reverse order. For a pair of primers to amplify a product one must be identical to the sense strand and the other identical to the minus strand or at the very least near identitiy and 3' complementarity of the primer in each case. The images below are examples of primer design from the Human CD40 gene, but illustrate the concepts surrounding primer specificity.
- Now go back to primer3Manager and repeat this for the reverse primer. Compare the matches having 100% complementarity for both primers and those that have 3' complementarity. If the primers are "good" the hits in common should only include HPV 6. Some of the sequences of HPV 6 will not have 3' complementarity because the region provided spans two open reading frames E5a and E5b.
- Other considerations for a diagnostic is possible host/patient nucleic acid sequences contaminating the sample so, Chimpanzee, pig, dog other more exotic sequences are of less relavance to the specificity of a diagnostic test but human would be important these would provide false possitivity. If the primer pairs did not satifactoraly meet the criteria, then one would return to identify new sequences and/or primers and repeat the process.
- Congratulations you have designed primers which could be used to detect HPV 6 infection and thus differentiate between HPV 6 and other types of human papilloma virus.
Real-time Quantitative Polymerase Chain Reaction (Real-Time qPCR) using TaqmanTM chemistry
The use of sybr green in a diagnostic setting offers some advantages oevr other PCR based chemistires for example simplicity, lower cost of the reagents whilst retaining the ability to quantify template concentration. However, Sybr Green based assays are prone to false positive signals associated with the poor specificty of the primers (mis-priming) or primer-dimer formation and end point melting curves are always performed following comlpletion of a such assays to confirm the absence of non-specific amplification. Other probe based assays such as Taqman offer greater level of specificity conferred by the additional requirement for target specific probe sequence internal to the primers. As a consequence mis-priming does not result in positivity due to non-complementarity of the probe sequence to the mis-primed product.
There are a number of different qPCR technologies in the practical we will only look at one of the common detection chemistries used often refered to as "Taqman". This historically was first commercialised by a company using the trade mark "Taqman". It is a technique which utilises a third oligonucleotide sequence internal to the primer pairs in PCR. The detection system used is the fluorescence released by the degradation of this "internal hybridisation" probe during the amplification process. In its undegraded state the probe has a fluorecent molecule attached to the 5' end which is excited by light of a specific wave length but passes the energy from this process through the molecule (termed forster resonance energy transfer or FRET) to another fluor at the 3', this energy transfer prevents the 5' fluor from emitting light with a specific spectra . The degradation of the probe by the polymerase during amplification releases the 5' fluor and causing it to emit light on excitation which can be detected by a photomultiplier. One of the additional attractions of this approach is assays can be multiplex ie multiple assays in one tube.
The amplification plot above shows a typical sigmoidal curve, characteristic of the increasing effect of multiple factors limiting the efficiency of the reaction with each cycle. The verticle axis represents the fluorescence obtained progeressively after each cycle (horizontal axis). This measure directly reflects the accumulation of product in the reaction and the rate of accumulation decreases and eventually ceases altogether.
Exercise 2. Primer Design for Real-Time qPCR using Primer3plus web interface
- Now for this exercise we will design primers and a "taqman" internal hybridisation probe to amplify the same region of HPV 6 which used previously indicated by sequence alignment to be specific to that virus type, but this time we will use a sequence spanning a larger segment covering the end of E5a and after the start of E5b. Having completed the task we will each give a breif verbal summary of our findings to the group.
- If you have closed the Primer3, open the primer3plus web interface. On the upper left there in the drop down box, it should say "Detection". Open the text file HPVseq2.txt by clicking on the hyperlink. Copy the entire sequence and paste it into the text box for primer3plus. Then return back to this page without closing the browser tab/window.
- First we need to change some settings for the software to perform the design. Click on the check box in the middle at the bottom of the form "Pick hybridization probe" and now we need to change the settings specific to the requirements of taqman chemistry. Click on
- the "General Settings" Tab in the box adjacent to "Primer Tm" set Min to 57, Opt to 60, Max to 63 and the Max Tm Difference to 2.0
- the "Advanced Settings" Tab click on the check box Ues Product Size Input ...... and next to Product size set Min to 70, Opt to 80 and Max to 120
- the "Internal Oligo" Tab : adjacent to Hyb Oligo Tm set Min to 67, Opt to 70 and Max to 73 (Note these are 10 degrees higher than the primer Tm settings
- Now take a look at the sequence and make some decisions about regions to exclude "< >" or to include this latter is identifed by the "[ ]" square brackets this defines for primer3 a target segment to be included within the amplified region. The advantage of this notation allows you to chose a region that avoids repetitive sequence or contiguous runs of the same nucleotide in a larger sequnce i.e. centering the design on a specific region. You mignt now want to "pick primers" and subsequently restrict the search by moving the include region or adding some exclude regions to the sequence.
- Beware that pressing the "primer3 back button" removes the annotation from the sequence and places it as numeric notation in the text boxes at the bottom of the form, pressing the browser back button retains this annotation. If you use the former it may be easier to remove the numeric descriptors and replace the include/exclude regions again. Any number of exclude regions can be given but only use a small single include region. The amplicon should be kept within the limits of our design criteria (see the lecture notes as a reminder). This is therefore a repetitive process each time adjusting the regions in order to obtain something acceptable.
- If you are satified next we need to make some manual checks specific to Taqman probe chemistry.
- Should not have more G’s than C’s If this is not the case we can make a note to use the reverse and complement of the probe sequence
- Avoid probes with runs of 3+ of the same nucleotide, especially G’s
- No Guanidines at the 1st or 2nd position at the 5’ end, these act as quenchers and since the nucleolytic degradation by the polymerase leaves the 5’ reporter dye attached to the first base and sometimes subsequent bases, such a molecule would provide a poor readout
- probe and primers anneal to the target, the 5’ end of probe should be as near to the 3’ end of the primer on same strand (max of 10-12). This tends to lead to more efficient displacement and degradation of the probe
- Specificity can be again checked using BLAST comparison via primer3Manager just as before and is particularly important for the probe sequence to ensure the signal obtained is specific to the target gene.
Summary (also see the notes at the start of the section and the glossary of terms below)