Sys Rev Pharm 2020; 11(2): 427 436 A multifaceted review journal in the field of pharmacy E-ISSN 0976-2779 P-ISSN 0975-8453 An Optimization and Common Troubleshooting Solving in Polymerase Chain Reaction Technique Shaden M. H. Mubarak1; Dhafer A. F. Al-Koofee2*; Ohood A Radhi3; Jawad Mohammed Ismael4; Zubaida Falih Al-Zubaidi5 1,2,5Dept. of Clinical Laboratory Science/Faculty of Pharmacy/University of Kufa 3 Faculty of Nursing/ University of Kufa 4 Dept. of Biochemistry/Faculty of Medicine / University of Kufa P.O. Box (21), Najaf Governorate, Iraq *Corresponding Author E-mail: [email protected] Article History: Submitted: 18.11.2019 Revised: 28.01.2020 Accepted: 29.02.2020 ABSTRACT Many genetic researches now relies on the study of variants in genetic Correspondence: material through different, diverse of universal Polymerase Chain Dhafer A.F.Al-Koofee Reaction (PCR) methods. In that context, we are putting a highlighted Department of Clinical Laboratory Science on the most important fundamental aspects of PCR technology, which Faculty of Pharmacy help researchers to clarify and reduce the majority problems and University of Kufa difficulties may face them in the laboratory work of any kind of PCR E-mail: [email protected] technology in general. DOI: 10.5530/srp.2020.2.63 Keywords: PCR optimization; primer design; melting temperature; PCR troubleshooting; GC-high; DMSO; genetics. @Advanced Scientific Research. All rights reserved INTRODUCTION should be neither high nor low to avoiding produce 1. Rules of PCR primer design insufficient primer-template hybridization and low PCR From a common molecular biology laboratory technique product in high temperature, and non-specific products is Polymerase Chain Reaction (PCR) that utilized to build caused by a high number of base pair mismatches in low up enough many copies of a definite section of DNA for temperature. The optimal Ta for a set primer pair can be further analysis in many species (Reece et al., 2011)(King, calculated as: Ta = 0.3 x (Tm of primer) + 0.7 x (Tm of 2010). It is depend basically on the availability of product) 14.9. Most primers require an annealing temperature between (50 65)°C. It is normally set at (3- Taq polymerase, template of DNA that want to amplify, 5)°C below the melting degree. four different nucleotides (A, T, G, and C) that are 4. GC Content: It represent a number of G's and C's in essentially structural units for nascent fresh DNA. In the primer as a percentage of the total bases and prefer another hand, the reaction mixture needs primers, and a have a content between (45-60)% for each primer. solution buffer to supply an unchanging pH (Holden et 5. GC Clamp: To promote specific binding at the 3' al., 2003)(Barnes, 1994). end of primer, pay attention to the presence of G PCR primers are short pairs oligonucleotides strand, or C bases within the last five bases of the primer hybridizing to reverse strands and contiguous the target taken in consideration no more than three G's or DNA segment of attention and assists as an initial site for C's residues due to the stronger bonding of G and DNA amplification (Rotmistrovsky, Jang, & Schuler, C bases. 2004). Successful good and appropriate primer pairs 6. Repeats: Generally, to keep away from design is crucial for all PCR-methods researches. The misprime should be no repeat consecutive of di- important idea designing of primers set to a particular nucleotide such as (ATATATAT) or four of same DNA required region for amplification, each primer bases like (AGGGG). should be anneal to the unique sequences in opposite 7. Stability of 3'-End: The last five bases from the strand, which agreement with heading for the (5' 3'- 3')direction (Chavali, Mahajan, Tabassum, Maiti, & and vice versa result in less false priming. Bharadwaj, 2005). In general, poorly designed primers 8. Template Secondary Structure: Mainly may amplify other, non-intended sequences, so it is as template secondary structure is important in much an art form as a science and preferred criteria designing primers and its stability depends on should be met as the following guidelines describe the their free energy and Tm . characteristics of a primer sequence: There are a few frequent troubles that occur during 1. Primer Size: The size had better be long sufficient for primer design involving: 1) Primer dimer due to binding acceptable specificity and small sufficient for binding primers together rather than template (Figure 1a) ; 2) simply to the pattern, and the optimal accepted size of Hairpin loops formation caused by self-annealing of primers is (18-25)bp. primers (Figure 1b). 2. Primer Melting Temperature (Tm): It is the temperature at which (50%) of the DNA two- stranded Observation will split to single-stranded. The best result produced with 1. Many of designed software are available to Tm range between (52-58)°C. Primer pairs Tm be within (1-3)°C of one another to ensure apply assist primer pairs designing. NCBI Primer three stages (denaturation, annealing and design tool extension) of traditional PCR. http://www.ncbi.nlm.nih.gov/tools/primer-blast/, Primer3plus 3. Annealing Temperature of Primer (Ta): It is the http://www.bioinformatics.nl/cgibin/primer3plus appreciation of the DNA-DNA crossbred constancy. Ta 427 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020 Dhafer A.F. Al – Koofee et al / An optimization and Common Troubleshooting Solving in Polymerase Chain Reaction /primer3plus.cgi/, and IDT particularity of the designed primer pairs in https://eu.idtdna.com/Primerquest/Home/Index order to escape from any related sequence are recommended websites for this purpose. pseudogenes or homologs. 2. It is important and beneficial to engage a blast on NCBI to verify for the intended quarry a b Figure 1: Familiar troubles that occur with primers design (a) Primer dimers (b) hairpin loop structure. 2. MATERIALS magnified. Approximately 10mM is sufficient Through the working in each experiment of PCR should for most PCR reactions. be taken some considerations that involve the following: Taq DNA polymerase which consider a 1. To avoid contamination of all reagents of the standard and suitable thermostable enzyme experiment, laboratory coat, goggle and gloves had for amplification of most types of PCR. In better be wore. In addition, it is required to have general, using up to 2.5U per reaction is Material Safety Data Sheets for a right management of recommended of Taq DNA polymerase in a equipment and harmful materials that used in whole volume reaction. In addition to experiments. sometimes even 0.1U can succeed in 20ul 2. Keeping all reagents secure on ice pail throughout reaction volumes(White, Chiang, Chow, & preparation of experiment and leave them to thaw Broker, 2003). fully before setting up a process. Building blocks of new DNA strands which 3. Mainly the reaction components playing an important are deoxynucleoside triphosphates (dNTPs) role in successful amplification protocol which that involve four basic nucleotides- (A, C, G, achieved by six critical factors. and T). It is significant to maintain each Template desired target for replication could concentration of four dNTP between10 -15 be any DNA resource, like genomic DNA However, the excessive amounts of (gDNA) and complementary DNA (cDNA) or dNTPs can slow or inhibit PCRs. even plasmid DNA. However, the variable A suitable buffer for PCR that occur in input amounts of DNA for amplification appropriate substance setting for action of governed by the complexity or composition. DNA polymerase. Frequently pH of buffer For example, up to 25 ng of gDNA may be between 8-9.5 and stabilized by Tris-HCl. enough as an initial amount, and on the other Most PCR buffer comes with 10X, and hand up to 0.5ng of plasmid DNA is sufficient composed from KCl ion (35-100)mM, Tris- in a (25-50) (200-500) HCl 100mM, MgCl2 (0.5-5.0)mM and gelatin microcentrifuge PCR tubes. 0.01%. (Table: 1) demonstrate most common A two set of appropriate upstream additive reagents recommended with final oligonucleotide primer (F) and downstream concentration that promote PCR. oligonucleotide Primer (R) designed Finally nuclease-Free Water to avoid any specifically to bind with opposite sequence of contamination that cause DNA degradation wanted goal gene or DNA section to be (Pelt-Verkuil EV, Belkum AV, Hays JP, 2008). Table 1: Most common additive reagent with their final concentration Reagent Final concentration Dimethyl sulfoxide (DMSO) 1 10% Glycerol 5 20% Formamide 1.25 10% Bovine serum albumin (BSA) 10 100 µg/mL Polyethylene glycol (PEG) 5 15% Betaine 0.5 - 2.5 M 4. Accessories and equipment on bench involve using 5. At the end of PCR experiment you have to evaluate the micro-centrifuge PCR tubes with their rack, set of product through using gel electrophoresis and comparing automated pipettes that dispense from (1-100) with standard DNA known molecular weight sizes(Pelt- thermal cycler. Verkuil et. al, 2008). 428 Systematic Review Pharmacy Vol 11, Issue 2, Mar Apr, 2020 Dhafer A.F. Al – Koofee et al / An optimization and Common Troubleshooting Solving in Polymerase Chain Reaction 3. Making up a Reaction Mixture freshly formed DNA molecules; that is to say, it influences Template of DNA the reliability of the enzyme. The ideal dNTPs levels rely For good amplification, an ideal DNA quantity should be on the size of the amplification products, the applied. Ideal DNA scales for this process are (0.001 1)ng concentration of MgCl2, and the concentrations of the of plasmid DNA or (1 10)ng of genomic DNA. Excessive primers (Keohavong, Wang, Cha, & Thilly, 1988). DNA can be giving unspecific products in the PCR process.