DNA and RNA are both composed of nucleotides. A nucleotide contains a base, a sugar and one to three phosphate groups. DNA is made up of the bases

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2 DNA and RNA are both composed of nucleotides. A nucleotide contains a base, a sugar and one to three phosphate groups. DNA is made up of the bases Adenine, Guanine, Cytosine and Thymine whereas in RNA Thymine is replaced by Uracil. The sugar is deoxyribose in DNA but ribose in RNA. 2

3 The nucleotides are connected by a covalent phosphodiester bond that forms between the phosphate group sitting on carbon number five (5 ) of the sugar of one nucleotide and the hydroxyl group of carbon number three (3 ) on the sugar of another nucleotide. The chain of nucleotides are extended by addition of nucleotides to the 3 end of the growing chain and the chain is thereby described to run in the 5 to 3 direction. DNA is composed of two DNA strands running in an antiparallell way. The two strands are held together by hydrogen bonds between the bases. A and T can form a basepair having two hydrogen bonds whereas G and C form a basepair with three hydrogen bonds. This results in a GC base pair having a higher bond energy and melting temperature. 3

4 DNA is copied (or replicated) by a large number of enzymes and molecules in vivo. This process is copied by the polymerase chain reaction (PCR) in vitro. DNA polymerase is the enzyme responsible for assembling a new DNA strand using another DNA strand as template. Primers are required to initiate this process. In PCR the primers define the region to be copied. 4

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7 A proper design of the primers is a requirement for a successful PCR. Both length and sequence are important parameters in primer design. 7

8 G base pairs with C on the other strand with three hydrogen bonds whereras A base pairs with T (U in RNA) with only two hydrogen bonds. It requires more energy (higher temperature) to break the higher number of hydrogens bonds found in a GC rich duplex. A longer duplex forms a higher number of hydrogen bonds and also requires higher energy. Positive ions (Na + ) from salt can neutralize the negative repulsions between phosphate groups in DNA and thereby stabilize DNA and raise the melting temperature. Primers with >5 C difference in T m may result in preferential amplification of the most efficiently primed product strand and lead to reduction in yield or even no amplification. 8

9 Basic Melting Temperature (Tm) Calculations The two standard approximation calculations are used. Both equations below assume that the annealing occurs under the standard conditions of 50 nm primer, 50 mm Na +, and ph 7.0. For sequences less than 14 nucleotides the formula is Tm= (wa+xt) * 2 + (yg+zc) * 4 where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively (from Marmur,J., and Doty,P. (1962) J Mol Biol 5: [PubMed]). For sequences longer than 13 nucleotides, the equation used is Tm= *(yG+zC 16.4)/(wA+xT+yG+zC). See Wallace,R.B., Shaffer,J., Murphy,R.F., Bonner,J., Hirose,T., and Itakura,K. (1979) Nucleic Acids Res 6: (Abstract) and Sambrook,J., and Russell,D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY. (CHSL Press) Salt Adjusted Melting Temperature (Tm) Calculations A variation on two standard approximation calculations are used. For sequences less than 14 nucleotides the same formula as the basic calculation is use, with a salt concentration adjustment Tm= (wa+xt)*2 + (yg+zc)*4 16.6*log 10 (0.050) *log 10 ([Na + ]) where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively. 9

10 The term 16.6*log 10 ([Na + ]) adjusts the Tm for changes in the salt concentration, and the term log 10 (0.050) adjusts for the salt adjustment at 50 mm Na +. Other monovalent and divalent salts will have an effect on the Tm of the oligonucleotide, but sodium ions are much more effective at forming salt bridges between DNA strands and therefore have the greatest effect in stabilizing doublestranded DNA, although trace amounts of divalent cations have significant and often overlooked affects (See Nakano et al, (1999) Proc. Nuclec Acids Res. 27: (Abstract)). For sequences longer than 13 nucleotides, the equation used is Tm= (41 * (yg+zc)/(wa+xt+yg+zc)) (820/(wA+xT+yG+zC)) *log 10 ([Na + ]) This equation is accurate for sequences in the 18 25mer range (Howley,P.M., Israel,M.F., Law,M F., and Martin,M.A. (1979) J Biol Chem 254: [Abstract]). OligoCalc uses the above equation for all sequences longer than 13 nucleotides. The following equation is reportedly more accurate for longer sequences (>50 nucleotides, ph 5 9). Tm= (41 * (yg+zc)/(wa+xt+yg+zc)) (500/(wA+xT+yG+zC)) *log 10 ([Na + ]) 0.62F The term (41 * (yg + zc 16.4)/(wA + xt + yg + zc)) adjusting for G/C content and the term (500/(wA + xt + yg + zc)) adjusting for the length of the sequence, and F is the percent concentration of formamide. For more information please see the reference: Howley, P.M; Israel, M.F.; Law, M F.; and M.A. Martin "A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes." J. Biol. Chem. 254, ,

11 The hydrogen bonds between G and C nucleotides contribute more to the melting temperature than the bonds between A and T. The GC content is an important parameter to take into account during primer design. Regions with low (<20%) or high (>70%) GC content should be avoided. 10

12 Polymerases require the binding of nucleotides at the 3 end of the primer to begin elongation, and because of this, any nonspecific binding at the 3 end will adversely affect amplification. Non specific binding that occurs at the 5 end of the primer does not necessarily adversely impact amplifications since polymerases cannot begin elongation until the 3 end binds. The maximum G value for the five bases from the 3 end is used for this calculation. For primers shorter than 30 base pairs, G is computed using the following equation: G = H T S H = enthalpy S = entropy T = the user defined melting temperature or T m When G is negative, the reaction is spontaneous. When choosing between various primer pairs, a lower (more negative) G value is favourable. The presence of G or C bases at the 3 end of primers (GC clamp) helps to promote correct binding at the 3 end due to the stronger hydrogen bonding of G and C bases. However, multiple G and C can form internal, non Watson Crick base pairs that disrupt stable primer binding. Generally, sequences containing more than three repeats of G or of C in sequence should be avoided in the first five bases from the 3 end of the primer, due to the higher probability of primer dimer formation and unspecific binding. 11

13 Primers should contain less than four complementary bases (to each other) especially at the 3 end. Complementarity between two primers, especially at the 3 ends, can lead to the formation of product artifacts arising from amplified primer dimers and primer oligomers. The concentration of primers is much higher than that of target DNA in PCR. Therefore, if the primers exhibit complementarity they may hybridize to each other and form homo dimers or hetero dimers. A self (homo) dimer is formed due to intermolecular interactions of the same primer. A cross (hetero) dimer is formed due to intermolecular interaction between the two primers. In order to detect cross dimers, the sense primer in 5 3 direction is compared with the antisense primer in 3 5 direction for homology. Avoiding primers with 3 overlaps is extremely important in multiplex reactions where multiple primers are used. 12

14 Self complementarity can lead to stable hairpin formation with just four GC base pairs in the stem and three bases in the loop. If oligonucleotides form hairpins by intramolecular hybridization, they are not available for hybridization to the target regions. Avoid stable hairpins having a melting temperature close to or above the melting temperature of the primers as use of these primers will lead to reduced yield. Hairpin formation can be determined using the mfold server at 13

15 The distance between the 5 ends of the two primers defines the length of the amplicon. In general, primers distanced < 2000 bases apart are used. This allows for sufficient amplification of the target region. Special polymerases or polymerase enzyme mixes are needed for amplification of long amplicons. 14

16 The choice of annealing temperature is one of the most critical factors for a successful PCR. Although it can be predicted, experimental optimization of this parameter is a common step when setting up a new PCR protocol. This is often done by evaluating the results of a gradient PCR where multiple annealing temperatures were tested. 15

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19 The design strategy is different for purposes Aand B (previous slide). For detection of the gene (A), any part of the gene can be amplified and primer can be match a sequence anywhere in the gene, also in the introns, but not too far apart. They also have to bind the coding and non codding strands, respectively and be directed towards each other. For detection of mrna, primers have to be placed in coding regions (exons) but not too far apart. They also have to bind the coding and non codding strands, respectively and be directed towards each other. In the experiment, single stranded mrna is first converted to double stranded cdna by reverse transcription and thereafter amplified by PCR. For cloning a gene, the complete protein coding part of the DNA must be amplified, from the start (ATG) to the stop codon (TGA, TAA or TAG). If there are no introns, genomic DNA can be used as template, otherwise cdna must be used to avoid cloning the introns. Avoiding the introns is crucial in protein expression in prokaryotic hosts as they cannot remove introns. Avoiding introns in eukaryotic expression host is a recommendation because the size genetic construct will be smaller. 18

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21 Primers are also used in other protocols and have to be designed according to other rules. 20

22 Primers can be ordered from many Life science lab chemical providers. Some details have to be given upon ordering. 21