PCR 실험 도중 유용한 글이 있어 스크랩한다.
Problems amplifying GC-rich regions? Problem Solved!
No, it isn’t you that’s the problem, and you’re certainly not alone if you’re having trouble amplifying GC-rich sequence and/or understanding why GC-rich sequences are causing such problems in the first place! Amplification of GC-rich sequences by PCR has been an irritant for scientists for decades! When we say “GC-rich” we mean ?60% of the bases are either cytosine (C) or guanine (G.) There are several options available, which alone or in combination may help you to deal with this problem, but first let’s look at why GC-rich sequences are more difficult to amplify.
The Cause of Your Problems
Problem 1. Stability: Thermal and Structural
GC-rich DNA sequences are more stable than sequences with low GC-content. For PCR, this means that the higher the GC content, the higher the melting point of the DNA. Under pressure, such as when exposed to heat, the GC-rich sequences can take far more abuse than GC-low sequences. While the possible role of meditation and yoga having a calming effects on these bases allowing them to take more abuse and still stick together has not yet been investigated, the impact of their structure has been. GC-rich DNA sequences are more stable than sequences with low GC-content, but, contrary to popular belief, the hydrogen bonds are not the primary reason for this stability. Stabilization is mainly due to stacking interactions called base stacking. There is some beautiful biochemistry and biophysics behind this and if you’re interested is a fantastic explanation as to why this stacking occurs.
Fun fact! This is why Thermus thermophilus, an extremophile, has a GC-rich genome, also regions of our genome (assuming of course that you are human) that need to been transcribed very often such as promoter regions of popularly transcribed gene, are AT-rich, like the TATA box- neat huh?!
Problem 2. Formation of Secondary Structures
This point is very much tied to the first point. When GC-rich regions form secondary structures, particularly hairpin loops, they’re very stable and so they stick around and accumulate. These don’t melt well at usual PCR denaturation temperatures. Additionally, primers used to amplify GC-rich regions have a tendency to form self- and cross-dimers as well as stemloop structures that can impede the progress of the DNA polymerase along the template molecule leading to truncated PCR products. GC-rich sequences at the 3’ end of primers can also lead to mispriming.
What a disaster!
Poof! *the sounds of your confidence in your ability to do PCR deflating*
But wait! We have some solutions for you!
Solution 1. Increasing Your Melting Temperature
This is a very sensible solution (in theory.) The higher the temperature, the more likely those troublesome secondary structures formed by GC-rich regions are to separate. However this results in lower product yields as your taq begins to denature more quickly at temperatures in excess of 92.5°C, so it is advisable to only use higher melting temperatures for the first few cycles and to avoid going over 95°C. It might take some playing around but this is solution is a good starting point.
Solution 2. Adjusting Your Magnesium Concentration
Non-specific amplification in general can be exacerbated and even caused by using excessive concentrations of magnesium (Mg) so test the optimum concentration using gradient or titration PCR. In a nutshell, your well on the absolute left contain a less than what you think will work and your well on the absolute right contain an excessive amount of magnesium with a gradient in the middle so you can determine the lowest possible amount you can use to achieve your product.
Solution 3. Calling upon a friend:
I found a few recipes online that various labs have found worked well for them:
- Addition of 3-10% dimethyl sulfoxide (DMSO), most often 5%
- Addition of 1M betaine and 5% DMSO – these may reduce your enzyme activity so add extra enzyme
- Addition of betaine (Sigma), DMSO, DTT and BSA
- Addition of ethylene glycol with 1,2-propanediol
- Addition of 5 % DMSO and 1.25% formamide or just formamide
- Addition of 5 – 10% absolute glycerol and higher temperature of annealing
- Use regular HotStart and add 2M betaine
- Use Kapa’s HiFi Hot Start enzyme
- Use KAPA3G GC rich Kit with 5% DMSO
- GC enhancers available in various kits like this Clontech, Epicentre and Qiagen also both offer kits for this problem if you’ve some money to spare.
Some of these options are beautifully summarized with links to references here. We also have a post detailing different PCR additives and how they can help you.
Solution 4. Other types of PCR
- Nested-PCR: I’ve written entirely on this technique so stay tuned for that!
- Slowdown-PCR: explored here.
- Hot-Start PCR.
- Long range PCR: clontech and qiagen both offer kits for this problem if you’ve some money to spare.
These forms of PCR and more are described simply and briefly here.
So you’ve looooads of options! Let us know what has worked for you in the comments below.
Base stacking: Kool ET. (2001) Hydrogen bonding, base stacking, and steric effects in DNA replication. Annu Rev Biophys Biomol Struct. 30:1–22.
Betaine and DMSO: Bhagya CH, et al. (2013) Polymerase Chain Reaction Optimization for Amplification of Guanine-Cytosine Rich Templates Using Buccal Cell DNA. Indian Journal of Human Genetics 19.1:78.
Ethylene glycol with 1,2-propanediol: Zhizhou, Z. et al. (2009)Enhanced Amplification of GC-rich DNA with Two Organic Reagents. BioTechniques 47.3 : 775-79. Web
Using formamide in PCR: Sarkar, G., et al. (1990). Formamide can dramatically improve the specificity of PCR. Nucleic Acids Research, 7465-7465.