https://www.researchgate.

net/post/What_exactly_is_the_function_of_DMSO_in_PCR

Difference between Universal Primers and Degenerate Primers?

n addition to António's answer, PCR/sequencing primers that bind to a sequence found in many plasmid cloning vectors, most
of which are derived from pUC vectors, are referred to as universal primer. These sequences are defined as good PCR and
sequencing sites as they flank the multiple cloning site. Universal primers are really not 'universal' in the sense that they will bind
to anything.

A degenerate primer is mixture of primers that has substitution of different bases sequence (they are similar not same). They
are usefull if need to amplify a gene from similar organism. So it possible amplify different sequence which represent different
protein sequence. They maybe used when need to design primer based on protein sequence. As we know different codon
codify for different protein.
Universal primer is a sequence (single) used for the amplification of a similar gene that related to a specific Genus. In bacteria
we need it to amplify ribossomal RNA. For example to amplify enterobacteriacea gene we need universal primer for it. That way
all species of enterobacteria are amplified (eg: Escherichia, Salmonella)
http://online.liebertpub.com/doi/pdfplus/10.1089/cmb.2005.12.431
http://bitesizebio.com/18992/a-primer-for-designing-degenerate-primers/

In addition to António's answer, PCR/sequencing primers that bind to a sequence found in many plasmid cloning vectors, most
of which are derived from pUC vectors, are referred to as universal primer. These sequences are defined as good PCR and
sequencing sites as they flank the multiple cloning site. Universal primers are really not 'universal' in the sense that they will bind
to anything.

vDegenerate primers
•It’s a combination of oligonucleotide sequences in which few bases are altered in such a way that the primer covers the all
possible nucleotide combinations for the targeted protein through the DNA sequence
•ATCGTT[GC]AAGT[AGC]ATC : GC= S, AGC=N
-Universal primers
•Anneal universally to organisms and amplify.
•conserved region is used
•Eg. Universal primers are used to amplify the 16SrRNA gene from the rumen metagenomic DNA (Weisburg et al., 1991), RNA
isolation

PCR
What exactly is the function of DMSO in PCR?
It decreases the Tm but does it have any other function during PCR. I tried my PCR with temperature gradient 1.5 degree
celcius less as calculated after adding DMSO, but my PCR did not work but when DMSO was added it worked.
The actual function of DMSO in PCR is it goes and bind to the DNA at the Cytosine residue and changes its conformation which
makes the DNA more labile for heat denaturation. this is the reason for the lowering of tm and increased GC region. since most
of the primers are GC rich, DMSO indirectly facilitates the annealing of primers to the template this enhaces the amplification.
DMSO is the organic addictive used in most PCR cases, in order to improve the amplification of GC rich regions which further
increase the amplification of the targeted sequences.
u can use albumine too
Ya, Low molecular amides, sulfones and other organic additives also works!
thanks Camilo but i tried all other PCR additives but they were not working and GC content was also 52% only.
but after failing in all the trouble shooting in last i added DMSO ,and it worked.
i dont know the exact reason.............
Kalaiarasi Sivaji , thanks but my primers GC content was Only 52%.
DMSO and other additives not only increase the GC rich regions but they alter the melting characteristics of strands which
enhance PCR amplification. And, each additives have their role in different PCR products. Some additives works better in
specific PCR reactions which fails for other reactions..

K has right, and what is the Tm of the primers, exist a diference of <10°C between them?
one primer is Tm is 62 and other ones is 63.5.
t decreases the nonspecific binding of primers on DNA template.....
Helps to prevent secondary structure from forming upon cooling from 95 C.
One among the failures of PCR reaction is secondary structure formations in either/ both the template or primers which lead to
showing multiple band patterns in your ran gel and some times no bands. One of the subsitute for this problem is adding
additives like DMSO, BSA, Glycerol etc..DMSO inhibits secondary structure formations in the DNA template or the DNA primers.
Hence your template DNA and primers will be in primary structure so that primers will easily bind to template. Generally will add
to PCR mix before reaction where minimizes the interfering reactions.
Here i am attaching general primer design protocol which gives you a good idea about secondary structure formations in
primers. I hope it may useful to you.
The actual function of DMSO in PCR is it goes and bind to the DNA at the Cytosine residue and changes its conformation which
makes the DNA more labile for heat denaturation. this is the reason for the lowering of tm and increased GC region. since most
of the primers are GC rich, DMSO indirectly facilitates the annealing of primers to the template this enhaces the amplification.
DMSO may also act on the enzyme : years ago it was used to relax sthe specificity of aminoacyl-tRNA synthetases
Friends Thank you all for your valuable suggestions....... thanks

I have a PCR with 3350 bp and has around 68% of CG. but I didn't get good PCR band yet. by gradient PCR I assign proper Tm
for my primer pairs. in these tempratures (lower than 53 degree of celcius) my PCR product (3350 bp) is faint with many extra
sharp non-specific bands. How can I improve my PCR? I want to try by additive like DMSO or betain.
Use of DMSO can also reduce certain Taq enzymes' activity up to 50% while reducing secondary structure of GC rich
templates; but it may not always help.
Betaine at ~1.2 M final concentration can stabilize primer-template duplexes in AT-rich regions and can improve product
specificity, although it, like DMSO, can reduce Taq activity as well.
But, to get more specific product, lessening Taq's activity is sometimes the right course of action; in addition to playing with the
thermoprotocol on the machine.
Also, too much Mg++ can be a problem; though it increases Taq activity, it does so at the expense of specificity.

Does anyone know a publication on this topic?

I used 5% DMSO with TaqMan and an 80% GC-rich sequence and it improved the results a lot. Adding more or changing
temperatures didn't help.
If gc content is more in primer go for dmso or glycerol or q solution of qiagen
if you have more GC content in your primers, you need to use DMSO, as it facilitates the annealing of primers to the template
then enhances the amplification process. TQ
Mohammad Arif Hossain
The Jikei University School of Medicine
I use it for exon 1 multiplication. Because exon 1 is always high gc rich. DMSO is very effective in pcr mixture for its easy
expansion

Manuel Otte
Institute for Clinical Microbiology
I use DMSO in some PCR reactions. It somehow increases the output of amplified DNA. I use 3% DMSO final concentration in
master mix.

Gabriela Montero-Morán
Universidad Autónoma de San Luis Potosí
Can u use DMSO in real time pcr??
Sampathkumar Ranganathan
Pondicherry University
Please mention the concentration of DMSO for 50 micro liter reaction mixture.
Krishna Chaitanya Pavani
Ghent University
DMSO can even enhance the concentration of DNA, in DNA extraction process
Priyanka Ghongane
Autolus
DMSO disrupts DNA strands and helps in strand separation
Digvijay Singh Yadav
Central University of Punjab
Do i've to lower the tm also after using 3% DMSO? and do i've to use DMSO again while carrying out the sequencing?
Sonalika Kar
NIMR
It helps to separate multiple bands
Manoranjan Kumar Jha
Centre for Cellular and Molecular Biology
ADDITION of DMSO or glycerol to the PCR to minimize secondary structures in the DNA template. i.e it reduce the chances to
form Hairpin structure, and decrease mt too.
Athithan Velan
Pondicherry University
It help to prevent non-specific primer binding. Improve PCR amplification.
Agnieszka Cudna
Institute of Psychiatry and Neurology
What can replace DMSO?
Hanane Djenane
University of Valencia
Hello!

please, I want to ask if the DMSO affect the sequencing of the PCR products! I use it because I had a weak bands .
Juliana Castrillon
Griffith University
High Agnieska Cudna

You can try with glycerol inset of DMSO

Murthy Chavali
University of Wuerzburg
try using glycerol

Davis Kuchaka
Kilimanjaro Clinical Research Institute
Hi Murthy,

What does glycerol do when added as an additive to the PCR reaction

Murthy Chavali
University of Wuerzburg
Glycrol is added in order to prevent the formation of secondary structures in DNA for example it may help in avoiding the
formation of hairpin loops, there by increasing the efficiency of PCR
Mohammed Sulaiman
Gombe State University
Special thanks goes to all that contribute and I find your answers beneficial...

NOW; I'm using cloning primer for amplification i.e. primer that contain the leader seq or buffer-zone, RS and gene specific
prmer seq, but the amplification result is weak. instate of getting >0.5 kb (>500bp) I end off getting 250bp or so. The Tm of the
primer is 59oC and I used gradient thermocycler with range of annealing temp 47oC up to 61oC, Annealing time 40sec and still
the amplification level is low. PLS WHAT IS THE SOLUTION ?
Jose Loyola
Universidad Nacional Agraria La Molina
Hello, I am using FIshF1 and FishR1 COI primers for amplifying Pseudoplatystoma punctifer DNA. Finally I should sequence the
PCR products. I want to know if DMSO do not affect the sequencing of the PCR products. Thank you for your help.
Diptesh Das
National Dairy Research Institute
Hello, I am using expression primers having restriction sites plus extra 4 base pairs, I have tried gradient PCR with Dream Taq
as well as Q5 DNA polymerase but there has been no amplification so far.. please suggest me how DMSO would help with
optimizing annealing temp. ? at which range of temp should I use? Tm according to neb.calculator is 57 and 56, at which I have
tried already.. but nothing seems to work
Mohammed Sulaiman
Gombe State University
I faced almost the same challenge, but never use DMSO !. Try annealing temperature between 49-56 oC, time (60 sec - 120
sec) and initial extension of about 2 min or so (this depends on the size of the gene U want to amplify). Then, reduce the cycle
numbers to 20 cycles or so. Tq
Alemu Tekewe
University of New Mexico
DMSO often improves amplification of GC-rich sequences
Masuma Akter
Korea Institute of Science and Technology
DMSO is used in PCR to inhibit secondary structures in the DNA template or the DNA primers. It is added to the PCR mix
before reacting, where it interferes with the self-complementarity of the DNA, minimizing interfering reactions. DMSO in a PCR
reaction is applicable with high GC-content(to decrease thermostability).
Pranay Mandal
Indian Institute of Science
What percent of DMSO to be used?
Masuma Akter
Korea Institute of Science and Technology
@ pranay Mandal ,I do not know exactly what is your purpose with DMSO. If it is investigating the Effects of DMSO on PCR
Fidelity, so you will find 5% of DMSO is effective.

Dhia Hussain Jassim Al-Delemi

University of Al-Qadisiyah
The actual function of DMSO in PCR is it goes and bind to the DNA at the Cytosine residue and changes its conformation which
makes the DNA more labile for heat denaturation. this is the reason for the lowering of tm and increased GC region. since most
of the primers are GC rich, DMSO indirectly facilitates the annealing of primers to the template this enhaces the amplification.
Radmila Janjusevic
The Cooper Union for the Advancement of Science and Art
I found this very intriguing: for many years and from time to time I am getting the emails saying that new answer was added to
this Q. So today I was curious and checked: this question was asked 5 years ago (which you can see by the time when first
answers were posted) and people are still adding the answers: with 18204 reads, I think without doubt Gopal Kushawah your
question wins the popular vote!