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QPCR oligo design

In DNA, PCR, Polymerase Chain Reaction on May 19, 2009 at 4:57 pm

Designing QPCR oligos might seem complicated but there are some rules and softwares that can make it easy. In our lab, majority of data points we generate are probably measured by QPCR, so I think  it is worth to review an algorithm for desiging QPCR assays.

So, today I will describe the way I design QPCR oligos. You can have a basic intro in PCR here.
More than a year ago I switched to the UPL system, the library of probes designed by Exiqon and now marketed by Roche. The concept is quite clear, the LNA modified oligonucleotides bind much stronger to the DNA template than the average oligonucleotides. By this we can decrease the lengths of them by keeping the Tm unchanged. The UPL library consists of 165 individual oligonucleotides that are in general nine basepair long and together cover the entire genome in respect of the coverage needed to design a QPCR oligo set for any gene. You can read more about the LNA nucleotides here and here. A good website where you can calculate the Tm of the LNA oligos here: http://lna-tm.com/. The UPL system is described here.

The system allows the design of an oligo set for any DNA sequence in the UPL Design Centre. You can access the Design Centre directly here.

UPL Assay Design Center

UPL Assay Design Center

Before we design an assay let us first look for the transcripts of a specific gene. It is very important to use annotated data, since in the annotated genomic data we have informations about possible SNP variations. This might be important, since the oligos and especially the probe should bind to an SNP free free region, because an SNP might disturb the binding of the probe to the template.

To make this data available we should use not the sequence but the transcript ID from the Ensembl.

Ensembl

Ensembl

At Ensembl select your species of interest, e.g. mouse and write the name of your gene of interest in the search box:

search box

If you write into the box a gene of interest (e.g. COUP-TF2) we will see the results as it is shown here:

couptf search

Here I have to click on the link of the gene and I will find the following screen: transcript infoThe most important info we are looking for is in the table on the top of the page:

transcripts

The two transcripts of the gene are:
ENSMUST00000089565
ENSMUST00000032768.

We will use these ID-s in the UPL Assay Design Centre. First select as organism the “Mouse”, and write into the box the two Ensembl transcript ID-s selected by commas. designIf you follow the steps the results will be like this:

resBelow this data you can see two links as it follows:common assaysTo design an assay that would measure all transcripts select: “common assays”.

The results will be given in a downloadable pdf report. Save this file and name is by the name of the gene you used as input.

The results in the pdf file look like this:

results

You can see that the amplicon is 95 bp long, there is no SNP in the binding regions of the primers and probes and the probe is closer to one of the primers. There is an SNP in the gene that was avoided by the program. You can have an SNP even in the amplicon, unless is not in the binding site of the primers or the probe.

Before I order the oligos, I usually test them with e-PCR on the UCSC Genome Browser.

UCSC Genome Browser

UCSC Genome Browser

Select the PCR view and paste the oligos into the given locations. Select the genome, the assembly and the target as “UCSC Genes”(If you used a genomic sequence for design use the target: “genome assembly”).

in silico PCRIf you have a hit, click on the link provided and the results will be represented in the genome as seen here:

browser results

The oligos are intron spanning and in the right location. Order the oligos in an HPLC pufied form in the lowest available scale for the first try. Be aware that according to the experience of several groups, and my own experience too,  only 2/3 of the UPL assays work without further optimization. This means, if you want to be sure from the first you better try two, three different assays for the same gene. The UPL Design Centre will generate several primer-probe sets and you can retrive these results too. Since the UPL library is given and one or two of the three ordered assays will work, it this worth trying three from the beginning!

In general the rules for a good QPCR assay:

1. The amplicon should be as short as possible (60-70 bp is ideal, but should be shorter than 100bp).

2. The Tm of the oligos should be around 60C, while the Tm of the probe 10C higher.

3. The distance between the the oligo and the probe should be as small as possible for a better exonuclease activity of the Taq polymerase.

4. The GC content of the two oligos should be as close as possible.

5. The number of GC bases in the last five nucleotides on the 3′ ends of the two primers should be identical (if possible).

6. Select for oligo sets with week internal bonds (less than four H bonds in the same conformation).

7. Avoid primer dimers that could produce artefacts due to the 3′ elongation of one of the primers.  The same for internal conformations.  Test it on the IDT webpage here. See below:conformations

8. Verify the oligos with e-PCR on the UCSC Genome Browser. The test should give one single hit!

9. If possible use annotated sequences to avoid the SNP effect.

10. If you are looking for genes (cDNA measurement) use exons that are common for all transcripts variants (or use the “batch assay-common assays” in the UPL Assay Design Centre)

Good luck!

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PCR or the Polymerase Chain Reaction

In animation, DNA, molecular biology, PCR, plasmid, Polymerase, Polymerase Chain Reaction, Replication, Taq on April 12, 2009 at 6:25 pm

When I first heard about the Polymerase Chain Reaction my first association was with the atomic bomb chain reaction. You know probably from your studies: the labile Uranium if receives a neutron it transformed to a stable Uranium isotope while several new neutrons are released. If these newly release neutrons meet novel labile Uranium atoms the reaction is amplified, more and more neutrons will be released until the system if is lost from control explodes in the form of an atomic bomb. If the reaction is under control we can produce heat and through this electricity in an electric plant, if the critical mass of the labile isotope is ignited with a neutron beam, it will explode.

You can have two excellent representations of the chain reaction below:

The chain reaction

But this is a blog about techniques in the molecular biology lab, so we will not deal with the fission chain reaction but we will see how a similar type of amplified reaction is produced with the DNA by specific enzymes in a reaction tube. The enzymes that can do a chain reaction are the Polymerases.

What is the function of polymerases? We have them in each of our cells. They do the most basic reaction that keeps life going on from the start of the very first organism ever. They are duplicating in a semi-conservative way the DNA in order to allow the transmission of the genetic information during cell division.

You can have an animation about DNA replication below.

What is the Polymerase doing?

The two DNA strands are connected by hydrogen bonds and code for the same information by the A:T and C:G base pairing.  The two strands are anti-parallel if we look to the double strand from one direction one of the strands will be in 5′-3′ direction and the other vice-versa in 3′-5′ direction. As an important rule we have to know that in nature all polymerases are doing the DNA synthesis in the 5′-3′ direction. The strand that can be replicated according to this rule is the leading strand. The other one is called lagging strand. The problem is that both strands have to replicated in by the same protein complex! But how can one single Replication complex produce the leading strand and the lagging strand in the same time ? We arrived to the problem of the the Okazaki fragments.

In the animation below you can find a good representation about how a single Replication complex can do the synthesis of the two different strands.

In order to speak about PCR we have to go out to trip to check some Geysers.

So let us check first the big Steamboat Geyser.

Yellowstone Steamboat Geyser

If we go closer to one of the hot springs we might see that the water is “living”, there are some algae, micro-organisms in this water. Let’s have a look:

The Yellowstone Hot Springs

These micro-organisms are living in really hot water. But if they are living, they should replicate, and if they replicate, they should have DNA polymerases!

These micro-organisms were isolated and one of them, called Thermo aquaticus (sometimes named Thermophilus aquaticus) became really famous. It has a polymerase that is used in vast majority of the in vitro DNA replication processes and in PCR.

I am sure you all have an idea already about PCR. We put all reagents needed for a DNA replication in a tube and reproduce the normal DNA replication process. So what do we need? We need a DNA template an oligonucleotide as a primer, the building blocks of the DNA (dATP, dTTP, dCTP, dGTP or in general dNTP -deoxi nucleotide tri phosphate), Mg and the Polymerase. If possible from Thermo aquaticus, which is called Taq.

There is one trick! This one trick was invented by Kary Mullis and he received Noble Prize for this single idea. The trick is, that we will not reproduce completely the natural reaction. We do not want to bother with leading strand and lagging strand and with all kind of Okazaki fragments and helicases and ligases.

The idea of Kary Mllis was that if you separate the double strand and design two oligos that will bind the two different strands but will look towards each other, than the product will be doubled. If you separate the strands by heating the solution to 95C you can repeat the reaction, and now you will have 4 copies. In the next run 8 copies and so on in each reaction you will have 2 on the power of the “cycle number” copies in a chain reaction fashion!!!

Let us have a really simple and good introduction to the whole procedure in the next two animations:

Below you can find a more fancy animation of the same procedure:

For this idea Mullis got the Noble Prize. His work changed completely the history of molecular biology. Let us check an interview with him about how he discovered PCR:

What is the practical use of this whole method?

Amplifying DNA by PCR became one of the most widely used method in a molecular biology lab. You can use it for transferring DNA from one plasmid to a different one, to introduce mutations and even to measure the amount of a specific gene in a sample. By combining it with a Reverse Transcription reaction we can measure copy numbers of RNA molecules.

Below we can see an example of how it is used in criminal justice!

In the next video you can see the workflow of DNA sequencing with a New Generation sequencing machine. What is remarkable here is that the designers of the instrument are skipping the cloning of the DNA fragments into plasmids and amplification of the plasmids by bacteria. They use micro reactors in the form of an emulsion PCR. One oligo is on a bead and the DNA binds the oligo. Each bead is fused with a small droplet that contains all the other reagents for the PCR. By this trick you will have a clonal amplification of the DNA fragments. One bead will contain on  type of DNA and you skipped all bacterial work. The result is that you can sequence the whole human genome in a couple of weeks for less the 100k USD. Or you can sequence a bacteria in a day…

Emulsion PCR in the FLX sequencer workflow

At the end of this lesson, lets have some fun and see the celebration of the PCR!