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Why should you use an annealing temperature about 5°C below the Tm of your primers?

Why should you use an annealing temperature about 5°C below the Tm of your primers?


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Why should you use an annealing temperature about 5°C below the Tm of your primers?

According to my current research, I think it has something to do with the other reactents in the PCR, but I am not sure.


You want to have primers which bind under the conditions of the reaction only to your sequence of interest. If you go too far off the optimal annealing temperature (5°C below the Tm is indeed a relatively good choice in my experience), your will affect the PCR efficency and thus the yield of your reaction.

An annealing temperature, which is much too low allows annealing of your primers to other sites than your intended target with partial annealing or internal base mismatches. This leads to unspecific amplification and lower yields.

If the annealing temperature is too high no primer binding can happen and you will get not PCR product. Alternatively, partial binding can also lead to misprimed products, although this is much less likely than getting no product at all.


The annealing temperature of a primer is defined as the temperature in which 50% of the nucleotides of the primer are bound to the DNA. 50% annealing proportion will not yield an optimal PCT product. Thus, by lowering the temperature by around 5oC, the proportion of bound primer changes to more then 50% (perhaps around 70 - 80%) thus getting a more precise yield.

This is why we subtract 5oC from the theoretical annealing temperature.


The importance of melting temperature in molecular biology applications

Learn how to predict and select appropriate melting temperatures for oligo hybridization steps, including PCR.

The melting temperature of an oligonucleotide duplex, or Tm, is the temperature at which half of the oligonucleotide molecules are single-stranded and half are double-stranded, i.e., the oligonucleotide is 50% annealed to its exact complement. Tm is a critical parameter to consider when designing and performing many molecular biology experiments, including PCR and qPCR.

Selecting primer melting temperature

Accurate prediction of Tm identifies duplexes that are likely to form at specific temperatures, allowing you to determine appropriate thermal cycling parameters. During the annealing phase of PCR, the reaction temperature needs to be sufficiently low to allow both forward and reverse primers to bind to the template, but not so low as to enable the formation of undesired, non-specific duplexes or intramolecular hairpins, both of which reduce reaction efficiency. Both primers in PCR should be chosen to have a similar Tm. IDT recommends selecting an annealing temperature 5&ndash7°C below the lowest primer Tm.

Selecting probe melting temperature

Designing qPCR assays with dual-labeled probes also requires careful coordination of primer Tm. When the reaction temperature is lowered from denaturing to annealing during cycling, the probe needs to anneal first to the target. If the probe binds to the target at the same time or after the primers bind, the polymerase may begin replication of target that does not contain bound probe. As a result, new DNA will be synthesized without associated probe degradation and, therefore, will not be detected as an increase in fluorescence. Such a situation leads to inaccurate data. For standard qPCR, IDT recommends a probe that has a Tm 5&ndash10°C higher than the Tm of the primers.

Double-check published Tm data

It is important to check the Tm of any oligonucleotide sequences used in PCR even when a previously successful primer and probe set is taken directly from a publication. The design of such published sequences may incorporate Tm enhancers such as a minor grove binder or locked nucleic acid bases. These Tm enhancers are not necessary for gene expression analysis unmodified probe and primer sets that provide reliable, accurate data can be designed for the same targets without the added expense of unnecessary modifications.

Predicting Tm

What is the best and easiest way to predict Tm? The OligoAnalyzer tool from IDT, found in the SciTools applications section of our website, is the result of continuing research and innovation. It takes into account the effects of oligonucleotide, cation, dNTP, and salt concentrations, oligonucleotide sequence, and nearest-neighbor interactions. The consideration of all these factors enables accurate prediction of oligonucleotide Tm specific to your reaction conditions [1]. For more information on the calculations and algorithm used by the OligoAnalyzer tool, refer to the IDT technical report, Calculation of Tm for Oligonucleotide Duplexes.


Protocol

Reaction Setup:

Add to a sterile thin-walled PCR tube:

Component 25 µl
Reaction
50 µl
Reaction
Final
Concentration
5X EpiMark ® Hot Start
Taq Reaction Buffer
5 µl 10 µl 1X
10 mM dNTPs 0.5 µl 1 µl 200 µM
10 µM Forward Primer 0.5 µl 1 µl 0.2 µM
(0.05-1 µM)
10 µM Reverse Primer 0.5µl 1 µl 0.2 µM
(0.05-1 µM)
EpiMark ® Hot Start Taq
DNA Polymerase
0.125 µl 0.25 µl 1.25 units/
50 µl PCR
Template DNA variable variable < 1,000 ng
Nuclease-free water to 25 µl to 50 µl

Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.

Transfer PCR tubes to a cycler and begin thermocycling:

Thermocycling conditions for a routine PCR:

Initial denaturation:
95°C 30 seconds

35-40 cycles:
95°C 15&ndash30 seconds
45&ndash68°C 15&ndash60 seconds
68°C 1 minute per kb

Final extension:
68°C 5 minutes


Primers TM - (Jun/24/2007 )

hi
on optimizing PCR products i use annealing Temp thats above or below the TM of the primer , but one of my collegues told me that i shouldnt be using annealing highr than the TM !!!!
is that true ? and why ?

Your annealing temperature should be below the tm.

the Tm is the temperature at which half your primers are annealed to their complementary strand (template). Above the tm, and you get less then half. And the fewer molecules you have annealed to the template, the lower you PCR yields will be.

It is true however that sometimes you get even better product with a annealing higher than primers Tm for some reasons.
And my colleague told me, that some assays even require higher temperatures to be working optimaly. But that is not a general guideline.
I usualy try classic 4 degs below Tm (as counted by primer3) as a first hit and then optimize further.
Set annealing in a range from lower temperatures to those close to Tm and if you see decline in the amount or quality of product, you simply choose the best temperature. But if you set this and product seems to be beter and better, then continue with higher temperature as you wish.
But generaly try only those under Tm.

My understanding was that it depends on the enzyme and the primer size. Also, there are several different ways to calculate the Tm. I go to the website of the primer I am using and calculate the primer Tm by their method, then use the product insert to determine the annealing temperature I will use.

Actually, that is a bit of an exaggeration- i actually just use my standard PCR cycle with an annealing temp of 53C, and if that doesn't work, then I start fiddling around with the annealing temp

Hi, the easiest way to find the optimal Annealing temperature is to do a gradient PCR. Sometimes , the optimal Annealing temperature can be higher than Tm. For example, the Tm of one pair of my primers was about 55C. But the optimal Annealing temperature was acutally 58.8C , as shown by the gradient PCR. So , I chose 58.8 as the annealing temperature and got very good results.

When temperatures under Tm are not working properly I also do a gradient PCR , I mean. I set the PCR to increase anealing temperature in each cycle. And most of the times it solve the problem.
But I also have a question. when you do this, how do you know which is the optimal TM. because the machine increase the temperature each time. It is not easy to find out about it?

I think what you are referring to is a touchdown PCR, which is different to a gradient PCR. With a gradient PCR you set each column of wells/positions in your PCR machine to a defined temperature, then when you run products on a gel to determine which one worked, you will have recorded what position in the machine that tube was- and that is your optimal temperature.

could u please tell me how to do gradient PCR ?
and what type of pCR device can do it?

I use a PCR cycle programed on our Biorad icycler that was created by another member of the lab so I don't actually know how to set one up from scratch. You could try consulting your machine's manual, or speak to a rep from the company who made it??


Primer Annealing Temperatures - (Aug/21/2012 )

I have two different annealing temperatures for my primers (59 and 55). At what annealing temperature should i set when in the PCR when using this primer set ?

I use this site to calculate the Tm :
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx
There are many algorithms around leading to different results. With the Tms determined here, I take the average - 5 oC for the annealing when I am using Taq polymerase (30 sec anealling step)
Take care that if you have overhangs (additional sequences you want to add to your sequence through PCR) you need to calculate Tm only for the matching part of the primers. For this purpose I use Phusion: higher Tm of both primers (matching sequence) + 5°C touch down -0.7°C / cycle 15 sec annealing step this for 14 steps followed by another 6 steps for which you have: lower Tm of both primers (calculate Tm for full sequence) (15 sec per step)

Too complex for me. I anneal at 55C.

Start with 55, if the product in unspecific increase annealing temperature by 2 degrees at a time. that should work

Step 1: anneal at 55
Step 2: (optional) do a gradient PCR annealing from 50-68
Step 3: Throw primers away, redesign primers.

Life is too short to deal with marginal primers.

I usually do..
Step 1: anneal at 60
Step 2: 5% DMSO
Step 3: forgot.. long time ago

why redesign primers? those are perfectly good primers. I did a PCR once with primers one at 46 and the other one at 71 (long story, but I couldn't design them better no matter what I did, and I struggled quite a lot). Worked perfectly. but I'm the one with a too complicated PCR program Designing primers with the same annealing temperature is so old-fashioned. What the PCR cyclers can do nowadays for you together with what we know about PCR can easily solve the problem of the PCR with different annealing temperatures. And btw: 4 degrees is nothing.

I am also all for the gradient annealing suggested by phage434. However, not all the labs invest in such a function of the PCR machine. (my current lab surely didn't invest in this option no matter how many times I cried for it ) But if your PCR machine can do gradient or touch-down: learn how to use them, they will save your life.

@ascacioc
Could you share the program you then used to get primers to work? I never violated the recomended 2 deg difference, even when I had some pretty awfull sequences too (but I work on nonextreme GC% templates), so seeing different approach would be interesting. Thanks.

I will post here the entire Phusion protocol I am using from mastermix to agarose gel:
1. Setup on ice in a 0.2 mL tube the mastermix for PCR according to the table
below. Thaw all non-enzyme components at RT, mix by short vortex and collect
by short centrifugation.
mastermix:
(75 &muL split into 6x 12.5 &muL)
-plasmid template
(sequencing or column grade
plasmid preparation)
x &muL (5 ng / 1 kb plasmid template)
-MilliQ H2O
(PCR quality = autoclaved in a bottle that was rinsed several times with MilliQ because you never know who made their Mn/Mg salt solution in that bottle before you once I have a bottle I can trust, I always reuse the same bottle for preparing PCR water all the time)
47.75 &muL - x &muL of plasmid template
-5x HF buffer 15 &muL
(final 1x)
-X_fwd primer
(5 &muM)
6 &muL
(final 400 nM)
-X_rev primer
(5 &muM)
6 &muL
(final 400 nM)
-dNTP-mix
(10 mM each)
1.5 &muL
(final 0.2 mM each nucleotide)
-Phusion
(2 U/&muL)
0.75 &muL
(final 0.02 U/&muL)

2. Split the mastermix into 6x 12.5 &muL and run all samples in a PCR cycler using
a 10°C temperature gradient (use columns: 1, 4, 6, 7, 9, 12 use the cycler
option menu to calculate the corresponding temperatures). (this is for the Eppendorf PCR machine the one for 96 samples with gradient included check for your own machine how it works)

PCR program ('' means sec and ' means min):
(20 cycles 105°C (works also with 99°C) heated lid and set to PAUSE)
ID &ndash initial denaturation 30&rsquo&rsquo 98°C
D &ndash denaturation 5&rsquo&rsquo 98°C
A &ndash annealing 15&rsquo&rsquo = higher Tm of both primers + 5°C
gradient +/-5°C
touch down -0.7°C / cycle
(calculate Tm for matching sequence)
http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx
E &ndash extension 15&rsquo&rsquo /1 kb 72°C
go back to step 2 and repeat 13 times
D &ndash denaturation 5&rsquo&rsquo 98°C
A &ndash annealing 15&rsquo&rsquo = lower Tm of both primers
(calculate Tm for full sequence)
http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx
E &ndash extension 15&rsquo&rsquo / 1 kb 72°C
FE &ndash final extension 3&rsquo 72°C go back to step 5 and repeat 5 times
S &ndash storage 8°C
3. Load all samples on a 0.8 % SB agarose-gel (7.5 V/cm 40 min) (for me SB buffer for running agaroses is the ideal buffer TAE is an ancient buffer developed because they did not know better than Tris at those times. But this is a completely different story . Check for optimal annealing temperature among your 6 samples and repeat PCR with that temperature if more product is needed.

BTW: when repeating PCR never use more than 20 uL per 200 uL tube.

This is called Phusion-never fail PCR and was developed by my supervisor during master thesis, Dr. Alexander Schenk. Enjoy you PCRs

Little note: since in my current lab I do not have a gradient PCR machine, I skip the gradient and it still works just use the middle temperatures as described in the protocol. Still my PCR never failed:)


Why should you use an annealing temperature about 5°C below the Tm of your primers? - Biology

Give me a P! P! Give me a C! C! Give me an R! R! What do yo get? Lots and lots of copies of DNA! I LOVE proteins &ndash so I talk most about those &ndash but to get to proteins you need an instruction guide, so DNA you must provide. All aboard the DNA Pol train, with a PCR(Polymerase Chain Reaction) survival guide to fill up your brain! PCR is another fundamental technique that&rsquos become crucial to modern biochemistry (and even modern medicine!) It allows us to make lots of copies of specific regions of DNA allowing us to look closer and answer questions like: &ldquohow is a frog related to a dog?,&rdquo &ldquois there a mutation in this gene causing this disease?,&rdquo and &ldquois this viral gene present, indicating infection?&rdquoIt also allows us to (after making lots of copies of a protein recipe) stick DNA into cells to get them to make proteins we want! So how does it work? (And how to get it to work if it doesn&rsquot want to&hellip)

The instructions for making proteins (long chains of amino acid letters that fold up into cool shapes and act as molecular workers) are written in a different biochemical language &ndash DNA. Stretches of DNA that hold &ldquoprotein recipes&rdquo are called &ldquogenes&rdquo and, since the genetic code is universal, you can stick that gene to &ldquoany&rdquo cell and that cell&rsquos ribosomes (protein-making machinery) will &ldquounderstand&rdquo it and make the corresponding protein. Similarly, if you know what genes an organism (Including a person) has, you can know what proteins they make, whether they have mutated versions of some genes, how they relate to other organisms.

So, DNA&rsquos really important and kinda like how you might love your IKEA table but don&rsquot have a particular fondness for the instruction manual, but you need it, in order to better understand proteins I often have to deal with their DNA instructions, and to do this I need to make lots of copies of specific stretches of DNA, and thankfully PCR provides a way! The discovery of PCR is attributed to Karry Mullis (working for a biotech firm called the Cetus Corporation) who got the first glimmers of it in 1983 and published it as a legit technique in 1985 (and in 1993 won the Nobel Prize for it).

As with basically all &ldquobreakthrough&rdquo scientific discoveries, he couldn&rsquot have done it with out the work of countless other scientists working before him. (and we couldn&rsquot do the work we do today without the work of him and others!).

For example, the stage was set in part by Arthur Kornberg, who won the Nobel Prize for discovering an enzyme (reaction speeder-upper) that could copy DNA, using a single strand of DNA letters (deoxynucleotides (dNTs)) as a template for linking up (polymerizing) &ldquoopposite&rdquo letters into a complementary chain (which can then be used as a template for recreating that original template sequence thanks to the 1:1 &ldquooppositeness&rdquo of DNA letters (more below). He called this enzyme DNA Polymerase (DNA Pol) and he could get it to make single complementary copies of DNA, but Mullis&rsquo contribution was using that DNA Pol to make lots and lots of copies.

And we still do. But we typically use super-hardy versions of DNA Pol that can withstand the high temps we cycle them through in the process I&rsquom gonna tell you about. And that choice of polymerase is just one decision you have to (I mean get to) make when you go to the DNA copy machine! So hopefully today&rsquos post will help you iron out your routine. I&rsquom going to start with an overview of how it works (so hopefully not too jargony/wonky) but then I&rsquoll get into some details for those who want them. (apologies in advance for formatting &ndash full-time grad student doing full-time lab stuff&hellip) So let&rsquos go!

DNA (DeoxyriboNucleic Acid) is the biochemical language our genetic info&rsquos written in & its alphabet consists of 4 deoxynucleotide (dNT) &ldquoletters,&rdquo A, T, C, & G which have a &ldquogeneric&rdquo part made up of a deoxyribose sugar with phosphate(s) hooked up on the &ldquoleft arm&rdquo (5&rsquo position) has a hydroxyl (-OH) group as a &ldquoleft leg&rdquo (3&rsquo position) & then the different letters have different &ldquonitrogenous bases&rdquo (bases) that stick off as a &ldquoright arm&rdquo &ndash these bases are the single- or double-ringed parts.

dNTs use their generic parts to link together through phosphodiester bonds to form long single stranded DNA (ssDNA) & 2 complementary single strands &ldquozip together&rdquo using their unique base parts (A across from T, C across from G) to form double-stranded DNA (dsDNA). This double-strandedness protects it from damage (the bases are facing in) and allows for easy copying since if you unzip it one strand can be used as a template for making another.

This &ldquounzip and copy&rdquo is what happens in replication &ndash before a cell divides, it needs to copy all its DNA (its entire genome) so that it can pass on a full set to each daughter cell, and it does this with the help of DNA Pol, which brings together the freely-roaming nucleotides, holding the right ones together & helping them link up, while rejecting the wrong ones (ones that don&rsquot complement each other (e.g. don&rsquot let an A bind a C!)

PCR (Polymerase Chain Reaction) is a way to carry out this process in a (really tiny) test tube & only copy a small section of DNA, which we specify by using PRIMERS. Primers are short pieces of DNA (oligonucleotides, or &ldquooligos&rdquo) which we design to &ldquobookend&rdquo our region of interest (AMPLICON). So, for each reaction we design and order different primers (thankfully they&rsquore cheap!)

We need these primers because DNA Pol can&rsquot start chain-building from scratch &ndash it needs to start from a short double-stranded stretch. This is just one of its limitations (but sometimes limitations can be good! You don&rsquot want cells copying DNA randomly!) Another limitation of its is that it can only copy DNA in One Direction (5&rsquo to 3&rsquo). Before we get too far, let&rsquos make sure we&rsquore NSYNC&hellip (90s girl, sorry!)

The letter-linking is &ldquogeneric&rdquo because it only involves the &ldquobackbone&rdquo parts that all the letters have (phosphodiester bonds involve the phosphate & hydroxyl merging) so you can link letters in any order (e.g. ATTACA or CAAATT). But the strand-zipping is specific because it occurs through interactions of the unique bases. So the &ldquoopposite&rdquo of ATTACA is TAATGA, which is different from GTTTAA. But, writing the opposites like this is a bit misleading because opposite direction you should be reading.

If you have ATTACA, and you stick the complementary letters across from it, you get this:

BUT &ndash dsDNA is ANTIPARALLEL &ndash this means that the strands are running in opposite directions (one is 5&rsquo->3&rsquo and the other is 3&rsquo->5&rsquo with the &lsquo pronounced &ldquoprime&rdquo and referring to whether the left arm (5&rsquo) or &ldquoleft leg&rdquo (3&rsquo) of that end&rsquos sugar is free). So

And we usually write sequences 5&rsquo to 3&rsquo, so the &ldquocomplimentary sequence&rdquo to 5&rsquo ATTACA 3&rsquo is 5&rsquo AGTAAT 3&rsquo

This may seem like a mere technicality, but it&rsquos really important in reality! Because DNA Pol can only copy DNA in one direction, 5&rsquo to 3&rsquo, and you always have to keep in mind which way the &ldquotrain tracks run&rdquo

Train tracks? This is another weird analogy of mine &ndash I like to think of nucleotides as train tracks and DNA Pol as a train. This train can only travel on double-stranded track, so it has to lay the track down ahead of it as it goes (and it knows what track to lay down by making it &ldquomatch&rdquo the other side of the track (e.g. if the next track across from it (on the other strand) is a T, lay down an A). In PCR, primers provide the starting stations for the train (since DNA Pol needs double-strandedness to start, it&rsquoll only start where you make it double stranded (but shorter than the other strand so there&rsquos still stuff to copy. Basically, you want something like this:

PCR is run in cycles of 1️⃣ MELT (heat up dsDNA to unzip strands) 2️⃣ ANNEAL (cool down slightly to allow primers to bind and 3️⃣ EXTEND (starting where primers leave off, add nucleotides complementary to template strand until you reach end of template strand). After the 1st cycle (where Pol goes till it runs out of steam or out of time), this end is determined by other strand&rsquos primer because DNA can only *copy* it cannot &ldquocompose&rdquo so it&rsquoll run off the track corresponding to the position that strand started being copied from in the 1st round. (easier to explain in pics)

You do this over & over 🔁 (30 or so times) to get lots of copies (each time you get 2X as many copies because each new strand becomes another template strand).

It might sound like a ton of work &ndash and for the molecules it is! &ndash but for us, once we get the reaction set up, the hardest part is just waiting for it to finish! Not for Mullis, however&hellip It&rsquos &ldquoeasy&rdquo these days because we have machines called thermal cyclers that do the rapid heating and cooling and heating and cooling and heating and cooling and&hellip

But these machines didn&rsquot come on the scene until 1987 (and of course not every lab could afford them, etc.) so in the beginning scientists would manually transfer the reaction tubes from one water bath to another over and over again.

The reaction to link nucleotides together (nucleotide polymerization) is the same regardless of whether it&rsquos happening in your cells or in the tube. It&rsquos also the same for RNA (but using a different Pol). but here we&rsquoll speak in DNA terms. So the reaction takes 2 deoxynucleotide triphosphates (dNTPs)(have 3 phosphate (PO₄³⁻) groups linked together) and links them up. And when it does so, it kicks 2 of those phosphates as a molecule pyrophosphate (PPi)

So, same process &ldquoin vivo&rdquo (in the body) and &ldquoin vitro&rdquo (in an artificial setting like a test tube), BUT in cells there are lots of helpers, whereas in PCR the process is stripped down to its bare necessities:

🔹TEMPLATE DNA: dsDNA containing the sequence you want to copy (amplify)

🔹PRIMERS: short ssDNA complementing the ends of amplicon (1 to serve as a start site for each strand)

🔹dNTPS: nucleotide building blocks (letters) to be added

🔹BUFFER: liquid combo of salts & pH stabilizers to keep everything happy

🔹Mg²⁺: magnesium cation (➕ charged molecule) to act as &ldquochaperones&rdquo to help shield phosphates&rsquo ➖ charge

One of the hardest parts of carrying out a biochemical reaction is often bringing reactants together & keeping them together long enough to interact. Why&rsquos this so hard? Molecules like to be free to move around (they want high ENTROPY) & they don&rsquot like to be tied down. Entropy refers to how many different &ldquostates&rdquo a molecule can be in (this can refer to being in different places or in slightly different shapes (e.g. bond rotated a bit). It&rsquos sometimes described as &ldquorandomness&rdquo or &ldquodisorder&rdquo because the more ways something can move the less likely you are to know exactly where they are at any time.

Getting nucleotides to link up is like getting a bunch of kids running around at recess to link up to form a really long 3-legged race. First, you have to get them to come over, then you have to get them to stay still long enough to convince them to link up, and then once they&rsquore linked up you have to make it &ldquofun&rdquo for them even though they can&rsquot run around anymore because their movement is restricted by being linked to the person next to them. And Pol has an even harder time because it has to get the kids to link up in a specified order!

So how can one little protein do all this? By giving the nucleotides something they want in return &ndash ridding them of a couple of their phosphates. The phosphates stick off the 5&rsquo end & they&rsquore basically really concentrated negative charges clamped together like a spring. PHOSPHATE (PO₄³⁻) has a central phosphorous(P) atom connected to 4 oxygen(O) atoms. It has &ldquoextra&rdquo electrons (e⁻) so it&rsquos ➖ charged. Like charges repel, so phosphates don&rsquot like to be next to each other. So it takes effort (in the form of energy (E)) to bring & hold phosphates together (like compressing a spring) &ndash so we call these bonds &ldquohigh energy&rdquo and when they&rsquore broken apart that E&rsquos freed to be used for other things like paying cost of linking nucleotides together.

So, even though nucleotide polymerization is still energetically costly because you&rsquore tying down molecules, ⬇️ their freedom (⬇️ entropy), this is compensated for by the large ⬆️ in entropy that occurs when PPi is released & hydrolyzed (split by water) to give you 2 individual orthophosphates (Pi) (and 2 little things moving around freely has even more freedom than the 1 medium-little thing (PPi) that&rsquos initially released &ndash so you get a double-boost

BUT in order to get this benefit, you 1st need to get them to react & this is often where proteins &/or RNA CATALYSTS (reaction speeder-uppers) called ENZYMES come in. They act as a sort of &ldquomediator&rdquo bringing the right reactants together, holding them in optimal positions to react, stabilizing reaction intermediates. & providing a friendly environment.

As I mentioned before, Arthur Kornberg discovered some enzymes that help mediate DNA copying: DNA POLYMERASES (DNA Pols). DNA Pol helps hold an incoming nucleotide (of the triphosphate variety) close to the growing chain it needs to be added to & in the right position. The 3&rsquo hydroxyl (OH) group then goes in for the attack! It latches on to the 1st phosphorus (P) group of the incoming nucleotide -> that P now has too many bonds, so it kicks out the other 2 phosphate groups as the inorganic phosphate molecule PYROPHOSPHATE (PPi) (energy boost 1). PYROPHOSPHATE is then hydrolyzed (broken by the addition of water) into 2 molecules of ORTHOPHOSPHATE (Pi) (energy boost 2).

DNA Pols have that general mechanism in common, but different organisms have slightly different DNA Pols that vary in EFFICIENCY, PROCESSIVITY, FIDELITY, & THERMOSENSITIVITY

  • EFFICIENCY: how fast can it go?
  • PROCESSIVITY: how many nucleotides can it add before it falls off template?
  • FIDELITY: how many typos does it make?
  • THERMOSENSITIVITY: how much heat can it take?

You can get higher FIDELITY (fewer typos) if your Pol has a &ldquoproofreading&rdquo 3&prime&rarr 5&prime exonuclease (DNA end-chewing) domain that can sense errors, &ldquobackspace&rdquo to remove them, & then put in the correct letter. This profreedng is important because errors will get copied&hellip & copied&hellip & copied&hellip BUT it slows down process so you get lower EFFICIENCY.

Other parts of DNA Pol proteins can also help out. A way to increase EFFICIENCY is by increasing PROCESSIVITY &ndash keep Pol on the template. Constantly falling off & hopping back on surely slows you down! Processivity-enhancing domains (a &ldquodomain&rdquo is just a protein &ldquosection&rdquo) or separate processivity-enhancing &ldquosubunits&rdquo bind dsDNA to help latch Pol on. But importantly they don&rsquot bind &ldquotoo tightly&rdquo & they can bind any sequence &ndash this allows Pol to stay on but slide along

BUT before you can copy strands you have to unzip them & this too is energetically expensive (like peeling apart 2 pieces of stuck together tape). You have to put in energy to give the DNA molecules more energy so they wiggle around more & the strands come apart.

In your cells, enzyme helpers called HELICASES help unzip them using chemical energy from ATP, but our &ldquobare-bones&rdquo PCR version doesn&rsquot have these helpers. Instead, we get the needed energy from HEAT. In the MELT step, we physically heat up the dsDNA so the strands come apart. And we have to get it REALLY hot! (

95°C or 200°F). Human DNA Pol would be pretty useless at this temp bc same heat that causes strands of DNA to come apart (yay!) can also cause proteins to unfold (eek!) (just like chains remain chains when you melt DNA (you don&rsquot break up strong covalent bonds), heat denaturation of proteins leaves you w/chains of amino acids)

Thankfully there are organisms called THERMOPHILES that have evolved to live in super hot environments (like near thermal vents in the ocean). They have super-strong proteins that can withstand high temps needed PCR. The &ldquoclassic&rdquo PCR Pol is Taq, which was discovered in 1976 and gets its name because it comes from the thermophilic bacterium Thermus aquaticus. Taq really made PCR possible &ndash before that, scientists trying to copy DNA in the lab were using a DNA Pol from e. coli bacteria. That DNA Pol couldn&rsquot take the heat, so after each heat step, they&rsquod have to add more! In fact, the first thermal cycler, named &ldquoMr. Cycle&rdquo was designed with an open system so you could keep adding more. https://bit.ly/2FoUFkD

So Taq was a major, crucial, discovery. But it&rsquos definitely not &ldquoperfect&rdquo &ndash Taq tends to make a lot of typos (low FIDELITY). Pfu DNA polymerase (from Pyrococcus furiosus) makes fewer errors (so higher FIDELITY). BUT it has relatively low EFFICIENCY. Could we do better?

It&rsquos hard to get all 4, but that hasn&rsquot stopped scientists from trying! Scientists can stitch together parts they like from different Pols to get Pol &ldquochimeras&rdquo w/enhanced functions. Our lab uses a chimera called Phusion Polymerase (not a paid endorsement, just what we use!) It&rsquos based off of a Pfu-like DNA Pol (w/proofreading capability for increased fidelity) fused to a small dsDNA-binding protein called Sso7 (from Sulfolobus sulfactaricus) which serves as a processivity-enhancing domain. It can add 1000 nucleotides (1 kb) in only 15 seconds w/few errors! So we time out the extension step accordingly (e.g. if we want to copy a 4kb segment, we&rsquoll set the extension step for 4×15=60s

So, DNA Pol was one choice we need to make, but there are others too&hellip.

Where what do you want your start & stop sites to be? Design primers to match &ndash but not just any sequence&hellip It&rsquos really important to design the stations (primers) carefully. Like many things in biochemistry, it&rsquos largely a matter of AFFINITY & SPECIFICITY

SPECIFICITY. We want the primers to bind ONLY where we want them to bind. Say you ask some friends to buy you a train ticket for a trip from Kansas City to NYC Is that Kansas City, Kansas? Or Kansas City, Missouri? Some friends might think Kansas, others might think Missouri & you end up with 2 types of train tickets..

Similarly, if your primer can bind at multiple sites on the DNA, you end up copying different stretches of train track giving you a mix of &ldquononspecific products&rdquo which show up as multiple bands on an agarose gel you use to separate the DNA pieces by size & visualize them: http://bit.ly/agarosegelrunning

When you design primers, you want to make sure they match your site of interest & ONLY that site. Like a computer password, the longer the sequence, the more likely it is to be &ldquounique.&rdquo If there are multiple occurrences of sequence you initially choose you might have to lengthen it to include more of the surrounding sequence (like saying &ldquofind a blue house w/a red house on the left & a green house on the right&rdquo instead of just saying &ldquofind a blue house&rdquo) You can use free software programs like NCBI BLAST or Primer3 to help you check for specificity & design good primers

BUT too long a primer and you can face other problems that lead to a &ldquoPCR TLDR&rdquo (too long didn&rsquot read)&hellip

🔹 PRIMER DIMERS: this is where the primer binds to itself instead of to your template. These can be self-dimers (where 2 &ldquostart stations&rdquo or &ldquostop stations&rdquo bind to themselves or cross dimers (a start & a stop)

🔹SECONDARY STRUCTURE: a single primer can fold up into &ldquohairpins&rdquo & bind itself

This leads to less primer available to bind template, so lower yield (less copies made) & DNA Pol can end up using primers as a (really short) template, amplifying primer &ldquoartifacts&rdquo instead of desired amplicon. And the high primer concentrations needed to prevent template-template zipping, make such primer pairing more likely because there are more primer fish & fewer template fish in the sea

Secondary structure in the *template* can also be a problem &ndash some regions of DNA are tightly wound up, making it hard to get to the site to bind (it&rsquos hard to build a train station in the middle of a mountain pass). You also want to avoid repetitive stretches (things like &ldquoAAAAAA&rdquo) because it makes it easier to &ldquoslip&rdquo & misprime

Typical primers are usually

20 nucleotides (nt) long, but it depends on experiment type, etc. There&rsquos free software available (like NCBI Primer-BLAST, Primer3, & AmplifiX) to help you design primers to fit your needs.

AFFINITY: We want the primers to have high affinity (attractiveness & stickiness) for the site we want them to bind so that they&rsquoll bind there stably & not fall off randomly during the annealing or extension steps. BUT we don&rsquot want the affinity to be too high or it won&rsquot come off during the melt steps

Affinity is largely dependent on the primer length (longer primers have more interstrand bonds working together to keep the strands glued shut) & &ldquobase composition,&rdquo When G&rsquos & C&rsquos are across from each other they can form 3 H-bonds. But when A&rsquos & T&rsquos are across from each other they can only form 2 H-bonds, so G-C pairs are stronger than A-T pairs. So a higher &ldquoG-C&rdquo content (typically given as a % of bases) means stronger binding. Ideal is usually

note: this is why origins of replication (ORIs) (where DNA strands come apart to be copied before cells divide) tend to be &ldquoA-T rich&rdquo because it makes it easier to melt them apart

Just like it&rsquos easier to pull off a piece of tape from the end than the middle, it&rsquos easier to pull of primers from the end, so you might want to put a&ldquoG-C&rdquo clamp at the 3&rsquo end (1 C or G) to help latch it on tight

A common measure used to calculate/report this affinity is the Tm (melting temperature). It&rsquos the temperature at which 1/2 the primer is bound &ndash the higher the temp, the more energy the molecules have & the harder it is to get the DNA to &ldquostay still&rdquo & bind. A high Tm means the affinity&rsquos high enough to hold down the wriggling DNA. Sp higher Tm, higher affinity. You typically want

60°C & you want the Tm of the 2 primers to be similar to one another (within

You want to know these Tms because they&rsquoll help you decide what temp to use for your annealing temperature &ndash they temperature you program the thermal cycler to be at during the primer-binding step of each cycle.

How do decide? The higher the temp, the more energy the DNA molecules have so it&rsquos harder for them to be &ldquotied down.&rdquo At higher temps, they have to really like their binding partner in order to sacrifice the freedom to move freely. But at lower temps, they have less energy, so they&rsquore less &ldquopicky&rdquo & more likely to &ldquosettle&rdquo for less-optimal pairing. As a result, if the annealing temp is too low, your primers can &ldquomisprime&rdquo & bind at the wrong sites giving you a mixture of &ldquononspecific&rdquo products.

So you want to choose a Goldilocks ANNEALING TEMPERATURE where the DNA molecules have enough energy to seek out their soulmate, BUT not so much that they can&rsquot &ldquotie the knot&rdquo once they find it. To help you choose, you can use free software to calculate the melting temperature (Tm) of the primers based on their length & sequence (since G&rsquos & C&rsquos bind each other more strongly than As & Ts (thanks to their 3rd H-bond), higher GC content increases the Tm. Common annealing temps are

55-80°C, and you want the Tms for both primers to be similar so that they both get to be Goldilocks-happy at the same time.

How long should the extension step be? Basically, each cycle, you need to give DNA Pol enough time to copy the region between the primers. So the optimal time depends on length of the sequence you want copied (AMPLICON SIZE) & the speed (efficiency) of the DNA Pol. The &ldquoclassic&rdquo Taq polymerase (a DNA Pol that can tolerate the high temps required) can add

360nt/min, but &ldquonewer models&rdquo (either from other organisms &/or mutated) like Pfu Turbo can go faster (

1000nt (1kb)/min)) So, for example, if you&rsquore using Pfu Turbo to copy something 2kb long, you&rsquod want to make sure your extension is a little over 2 minutes. This is per cycle so with longer times, be prepared to do some waiting (a great time to pour that agarose gel you&rsquoll use to check that it worked!)

Just how long you&rsquoll have to wait depends on how many cycles you choose. And that depends in part on how many copies you want made (each cycle you increase # of copies exponentially (e.g. 2, 4, 8, 16, 32, 64&hellip), so by 35th cycle you&rsquod have 68 billion copies! You might think, the more the better, right? BUT the more copies you make, the more chance for errors to occur (& be copied) Common cycle #s are

25-30 (but even if you have to wait you don&rsquot have to manually move the tube over and over!)⠀


Why should you use an annealing temperature about 5°C below the Tm of your primers? - Biology

Policies For PrimerBank Usage

PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). There are several ways to search for primers: by GenBank Accession, NCBI protein accession, LocusLink ID, PrimerBank ID or Keyword (gene description). PrimerBank contains about 180,000 primers covering most known human and mouse genes.

Polymerase Chain Reaction Amplification (PCR) is one of the most actively used techniques in molecular biology. In recent years, PCR has been increasingly used for gene expression detection or quantification. It is a more convenient method in gene expression studies comparing to other techniques, such as Northern Blot. One common problem in PCR is the non-specific amplifications of other gene products because cDNAs libraries of thousand of genes are often used as PCR templates. Therefore, we need to carefully design PCR primers that specifically amplify the genes of interest. Unfortunately, most available primer design programs only focus on primer chemical properties, such as melting temperature, GC content, secondary structure, etc. Little emphasis is given to primer mispriming to other genes. In contrast, all primers in PrimerBank were carefully designed to ensure gene specificity.

The primer design algorithm has been extensively tested by real-time PCR experiments for PCR specificity and efficiency. We have tested 26,855 primer pairs that correspond to 27,681 mouse genes by Real Time PCR followed by agarose gel electrophoresis and sequencing of the PCR products. The design success rate is 82.6% (22,187 successful primer pairs) based on agarose gel electrophoresis.

You may search PrimerBank by GenBank Accession, NCBI protein accession, LocusLink ID, NCBI gene symbol or PrimerBank ID.

Search terms are automatically combined if they are separated by space. For example, search "kinase s6" returns all records with both keywords "kinase" AND "s6". AND and OR Boolean operators may also be used. Here are a few examples:

Keyword Interpretation
kinase s6 Searching for both kinase and s6
kinase and s6 Searching for both kinase and s6
Cytochrome or cyp Searching for either Cytochrome or cyp

Currently, only a limited set of keywords (keywords defined in the protein definitions) can be searched. For example, you cannot search by gene symbols. If you did not find your genes, please look up the corresponding gene IDs (DNA accessions, proteins accessions, or LocusLink IDs) and search by IDs.

Because of the sequence redundancy in GenBank, each gene is usually represented by more than one NCBI record. PrimerBank sometimes uses the NCBI LocusLink index file to map multiple sequence records to the same gene locus. As a result, a different accession number other than originally submitted may be retrieved. However, both accessions represent the same gene. If a retrieved record has a different GenBank accession, a LocuLink ID will appear in the gene description field.

You may use your browser's save function to save the web page in your local computer. Other saving options will be implemented later.

All the primers in PrimerBank were designed using a program called uPrimer. Great care has been given to avoid primer mispriming to other known genes in a genome. Here is a list of criteria for gene specific primer design:

  1. The primer length range: 19 - 23 nt, with the optimal length at 21 nt.
  2. The primer GC percentage range: 35% - 65%.
  3. The delta G value for the five 3&rsquo end-bases is at least -9 kcal/mol.
  4. The primer Tm range: 60 - 63 °C, determined by the Nearest Neighbor Method.
  5. The PCR product length range is 100 - 250 bp. If this requirement cannot be satisfied, alternative ranges will be used.
  6. The default number of primer pairs designed for each sequence is 3.
  7. No primer is designed from low-complexity regions.
  8. A primer does not contain 6 or more contiguous same nucleotides.
  9. A primer does not contain any ambiguous nucleotide.
  10. No repetitive 15-mer from other gene sequences in the genome (for both strands) anywhere in a primer.
  11. No repetitive 13-mer from non-coding RNA sequences (for both strands) anywhere in a primer.
  12. The global BLAST score for any primer is less than 30 (equivalent to 15-mer perfect match).
  13. The maximum Tm for the 3&rsquo end perfect match to other gene sequences does not exceed 46 °C does not exceed 42 °C when compared to non-coding RNA sequences (Tm determined by the Nearest Neighbor Method).
  14. For primer secondary structure (the primer-primer self-annealing)
    1. No repetitive 5-mer is allowed anywhere when a primer sequence is compared to its complementary strand.
    2. The four 3&rsquo-end bases should be unique when compared to the primer&rsquos complementary strand.
    1. No repetitive 9-mer is allowed when a primer sequence is compared to the complementary strand of its cognate sequence.
    2. The BLAST score is less than 18 when a primer sequence is compared to the complementary strand of its cognate sequence.

    All the primers in PrimerBank have melting temperatures (Tm) of 60 - 63 °C. A higher annealing temperature results in more specific priming. Previous studies indicated sufficient priming should occur at primer Tm. Therefore, an annealing temperature of 60 °C is recommended for all PrimerBank primers. Non-specific PCR products are likely to occur at lower annealing temperature. We have tested a few hundred primers under 60 °C annealing temperature and the PCR experiments worked very well.

    The primer Tm values are calculated using the Nearest Neighbor Method with the up-to-date thermodynamic parameters. Tm values are also dependent on primer and salt concentrations. Higher concentrations of primer and salt usually lead to higher Tm. The Tm values included in PrimerBank are for 0.25 uM primer, 1.5 mM Mg2+, 50 mM Na+, and 0.8 mM dNTP, which are typically used in PCR experiments. Other common PCR conditions only affect the Tm slightly.

    To guarantee PCR efficiency, small PCR products are recommended. Most PrimerBank primers lead to amplicons in the size range of 100 - 250 bp. PCR efficiency is close to 100% in this range (supported by real-time PCR experiments). However, PCR efficiency may be reduced for much larger PCR products. Less than 1% of PrimerBank primers lead to >400 bp amplicons. Please be cautious about PCR efficiency when these primers have to be used.

    Sometimes it is desirable to select a primer pair that spans intron. In this way, genomic DNA contamination can be closely monitored. Usually a PrimerBank primer pair spans intron because a typical exon is quite small. The primer intron-spanning information will be included in the next version update. If it is important for you to be sure that a primer pair spans intron, you may simply do a BLAST search of the primer pair sequences, or better the amplicon sequences (listed in the primer detail page) against the genomic sequences.

    Agarose gel electrophoresis or melting curve analysis may not always reliably reflect PCR specificity. From our experience, bimodal melting curves are occasionally observed for long amplicons even when the PCRs are specific. The observed heterogeneity in melting temperature was due to internal sequence inhomogeneity (e.g. independently melting blocks of high and low GC content) rather than amplicon contamination. On the other hand, for short amplicons very weak bands migrating ahead of the major specific bands are occasionally observed on agarose gel. These weak bands are super-structured or single-stranded version of the specific amplicons in equilibrium state. Although gel electrophoresis or melting curve analysis alone may not be 100% reliable, the combination of both can always reveal PCR specificity in our experience.

    Although all PrimerBank primers are designed to be gene-specific, we still need to be very careful about PCR conditions.

    Non-specific primer extension of only a few bases at low temperature by DNA polymerase can easily lead to non-specific PCR amplifications. Therefore, hot-start PCR is STRONGLY recommended. In fact, I only do hot-start PCR in my experiments. I routinely use AmpliTaq Gold polymerase (Applied Biosystems) for hot-start PCR.

    The annealing temperature may affect PCR specificity. To avoid non-specific PCR products, a high annealing temperature (the smaller one of the two Tm values from the primer pair) and a short annealing time are recommended.

    If you still see non-specific bands, it could mean the primer pair in use is the problem. Although great care has been given to design PrimerBank primers, the success rate is not 100%. Less than 1% of the primers may have design problems (see our paper for detailed discussion). In this case, please try a different primer pair for the same gene. Click here if you would like to report primer problems.

    Poor quality of PCR templates, primers, or reagents may lead to PCR failures. First, please include appropriate controls to eliminate these possibilities.

    Some genes are expressed only in certain tissues. Please first read literature to make sure your genes are included in the cDNA templates. In our experience, this is the most likely cause for negative PCR results. If you are sure the genes are expressed, then try lowering the annealing temperature (to Tm - 5 °C) and increasing the annealing time to ensure sufficient primer annealing.

    If you still could not see any PCR band, it could mean the primer pair in use is the problem. Although great care has been given to design PrimerBank primers, the success rate is not 100%. Less than 1% of the primers may have design problems (see our paper for detailed discussion). In this case, please try a different primer pair for the same gene. Click here if you would like to report primer problems.

    The algorithm and initial testing of PrimerBank were generated by Wang and Seed, Xiaowei Wang and Brian Seed (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Research 31(24): e154 pp.1-8. and further refinement and validation of the entire mouse collection was carried out by Spandidos and coworkers. Athanasia Spandidos, Xiaowei Wang, Huajun Wang, Stefan Dragnev, Tara Thurber and Brian Seed (2008) A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance. BMC Genomics 2008, 9:633


    Why should you use an annealing temperature about 5°C below the Tm of your primers? - Biology

    The polymerase chain reaction (PCR) is a biomedical technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes the diagnosis of hereditary diseases the identification of genetic fingerprints (used in forensic sciences and paternity testing) and the detection and diagnosis of infectious diseases.

    The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, after which the method is named, are key components to enable selective and repeated amplification.

    Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis.

    A basic PCR set up requires several components and reagents. These components include:

    • DNA template that contains the DNA region (target) to be amplified.
    • Two primers that are complementary to the 3′ (three prime) ends of each of the sense and anti-sense strand of the DNA target. or another DNA polymerase with a temperature optimum at around 70°C.
    • Deoxynucleoside triphosphates (dNTPs, sometimes called “deoxynucleotide triphosphates” nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand. , providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. cations, magnesium or manganese ions generally Mg2+is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis
    • Monovalent cationpotassium

    Procedure

    Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps. The cycling is often preceded by a single temperature step at a high temperature (>90°C).

    Initialization step(Only required for DNA polymerases that require heat activation by hot-start PCR): This step consists of heating the reaction to a temperature of 94–96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1–9 minutes.

    Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98°C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

    Annealing step: The reaction temperature is lowered to 50–65°C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. This temperature needs to be low enough to allow for hybridization of the primer to the strand, but high enough in order for the hybridization to be specific, i.e. the primer should only bind to a perfectly complementary part of the template. If the temperature is too low, the primer could bind imperfectly. If it is too high, the primer might not bind. Typically the annealing temperature is about 3–5°C below the Tm of the primers used. Stable DNA–DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation.

    Extension/elongation step: The temperature at this step depends on the DNA polymerase used. Taq polymerase has its optimum activity temperature at 75–80°C, and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5′ to 3′ direction, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified.

    Final elongation: This single step is occasionally performed at a temperature of 70–74°C (this is the temperature needed for optimal activity for most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

    Final hold: This step at 4–15°C for an indefinite time may be employed for short-term storage of the reaction.


    2.1: Amplify aptamer-encoding DNA

    Back when he was a postdoctoral fellow, Professor Niles screened a random library of RNA aptamers to find one that binds to heme &ndash the iron-containing site in hemoglobin. It is known that heme can bind to certain transcription factors and modulate gene expression (see references Zhang and Hach, 1999 and Ogawa, et al., 2001), and RNA aptamers are one potential tool for learning more about signaling networks involving heme. To select heme-binding aptamers, Professor Niles ran a pool of RNAs with 50 randomized base pairs through a heme affinity column. He then amplified the column-selected pool of aptamers and repeated the process several times. An aptamer called "6-5" survived through the 6th round of screening, but ultimately was found not to bind to heme. An aptamer called "8-12" survived through all 8 rounds of screening, and has a heme binding affinity of 220 nM. Both were described in the Niles, et al., 2006 paper referenced below.

    Today you will be given two archival plasmids containing the 6-5 and 8-12 sequences, respectively. RNA is not very stable compared to DNA thus, RNA aptamers are copied into their associated DNA sequences for long-term storage. Ligating the DNA fragment into a plasmid that can be carried in bacteria provides further amplification and storage capabilities. We will make use of these capabilities more extensively in Module 2.

    Figure (PageIndex<1>): PCR schematic. Depicted are two complementary strands of DNA, with a desired target fragment shown in green. Primers that can select the target sequence are shown as short arrows, with the dotted lines indicating the extension step of PCR. Note that in the first couple rounds of PCR, products longer than the desired target will be made (dotted lines keep extending). However, these early products themselves become templates that produce the correct product in abundance.

    In order to select and amplify just the short DNA fragment that encodes for the aptamer, you will use the polymerase chain reaction, PCR. PCR comprises three main steps: 1) template DNA containing a desired sequence is melted, 2) primers anneal to specific locations on the now melted (i.e., single-stranded) DNA, and 3) the primers are extended by a polymerase to select and create the desired product. Extension occurs at

    95°C, and annealing at a temperature

    5 °C below the primer melting temperature thus, the repetition of these steps is called thermal cycling. After each cycle, the newly formed products themselves become templates, causing exponential amplification of the selected sequence. (Note that the early rounds of PCR will not produce the desired product - we will see why in today's pre-lab lecture.)

    Once the PCR is running, you will begin to explore some computational tools for RNA analysis. During this module, you will ultimately use three different programs to explore both sequence similarities among RNA candidate aptamers and higher-order structures that arise from the primary sequences. For today, you will look at degrees of sequence similarity among a list of aptamers, some of which bind to heme and some that don't.

    Figure (PageIndex<2>): (Photo by Mark Robert Halper. Courtesy of Kary Mullis. Used with permission.)

    Based on the numerous applications of PCR, it may seem that the technique has been around forever. In fact it is only 25 years old. In 1984, Kary Mullis described this technique for amplifying DNA of known or unknown sequence, realizing immediately the significance of his insight.

    "Dear Thor!," I exclaimed. I had solved the most annoying problems in DNA chemistry in a single lightening bolt. Abundance and distinction. With two oligonucleotides, DNA polymerase, and the four nucleosidetriphosphates I could make as much of a DNA sequence as I wanted and I could make it on a fragment of a specific size that I could distinguish easily. Somehow, I thought, it had to be an illusion. Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and I would surely have heard of it. We would be doing it all the time. What was I failing to see? "Jennifer, wake up. I've thought of something incredible." --Kary Mullis from his Nobel Lecture, December 8, 1983.


    Conclusions

    Because the design of perfectly matching universal primers remains an unsolved problem ( Forney et al., 2004), the investigation of PCR parameters that could attenuate the quantitative bias associated with preferential amplification caused by primer mismatch is very important. Newly designed domain-specific primers are usually tested with genomic DNA of strains and environmental samples for PCR amplification efficiency ( Marchesi et al., 1998 Baker et al., 2003), but preferential amplification should also be considered and tests carried out to determine the importance of this phenomenon. PCR optimization strategies for the assessment of environmental communities by ‘universal’ primer sets are directed to increasing the specificity of amplification using relatively high annealing temperature ( Hansen et al., 1998) or ‘touch-down’ PCR protocol ( Simpson et al., 2000). The results presented here suggest the opposite strategy for reducing preferential amplification using specific but low-stringency amplification. (1) PCR amplification should be optimized to reach the lowest annealing temperature, where the reaction is still specific and unspecific products (mispriming) are not observed. The cycle number can be high at this step. (2) PCR amplification should be repeated at the optimal temperature using parallel samples at low cycle numbers (around 25). In the case of low yield, parallel runs can be combined to obtain sufficient quantities for subsequent analyses.



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