Nucleic Acid Design Applications Polymerase Chain Reaction (PCR)

1. Nucleic Acid Design Applications Polymerase Chain Reaction (PCR) Calculating Melting Temperature (Tm) PCR Primers Design

2. When Science named PCR and the polymerase that it employs as its first "Molecule of the Year" in 1989, the editor, Daniel Koshland Jr., provided a succinct explanation of PCR. He wrote: "The starting material for PCR, the 'target sequence,' is a gene or segment of DNA. In a matter of hours, this target sequence can be amplified a million fold. The complementary strands of a double-stranded molecule of DNA are separated by heating. Two small pieces of synthetic DNA, each complementing a specific sequence at one end of the target sequence, serve as primers. Each primer binds to its complementary sequence. Polymerases start at each primer and copy the sequence of that strand. Within a short time, exact replicas of the target sequence have been produced. In subsequent cycles, double-stranded molecules of both the original DNA and the copies are separated; primers bind again to complementary sequences and the polymerase replicates them. At the end of many cycles, the pool is greatly enriched in the small pieces of DNA that have the target sequences, and this amplified genetic information is then available for further analysis."

3. PCR Concept: Amplification of a relatively short piece of DNA for manipulation or sequencing. Driving phenomena of PCR: Heating and Cooling Heating: Double-stranded DNA “comes apart” when heated to near boiling. This is also called “denaturing” or “melting”. Cooling: Complementary DNA “comes together” when cooled. This is also called “renaturing”, “annealing” or “hybridizing”. Double-Stranded DNA COOLING HEATING Single-Stranded DNA

4. Molecular Basis of PCR: Polymerase Activity A Polymerase is an enzyme that synthesizes DNA. 1) DNA can ONLY be synthesized using the complementary strand! 2) Polymerases synthesize DNA in the 5’ 3’ direction! 5’-GTCGATGTCTGATCAATTGGGCTGATCATGTCGATGATGCTAGAAT-3’ 3’CTACGATCTTA-5’ 5’-GTCGATGTCTGATCAATTGGGCTGATCATGTCGATGATGCTAGAAT-3’ ACTAGTACAGCTACTACGATCTTA-5’

5. PCR uses the following reagents to AMPLIFY sections of DNA… • DNA template • Polymerase • Free Nucleotides (which are incorporated during DNA synthesis) • PCR Primers • Primers are two short pieces of DNA (each with a unique sequence) that are complementary to the two different strands of the DNA template. • In line diagrams, the primers are designated as arrows, where the arrows point in the direction of 3’ DNA synthesis.

6. This section of the DNA template will be amplified. Double-Stranded DNA HEATING 3’ 5’ 5’ 3’ PCR Primers Single-Stranded DNA

7. Double-Stranded DNA 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ HEAT (95ºC, 30 seconds) Single-Stranded DNA 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ COOL (60ºC, 30 seconds) PCR Primer Annealing 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCCTCCCCCACCGACCCCA-5’ 5’-GGATGGAACACTGGGGGGA-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’

8. HEAT (72ºC, 30 seconds) Polymerase Elongation 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ CTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGA 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ DNA Synthesis after 1 “cycle” of PCR = 1 double stranded DNA is now 2 “copies” 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’ 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGT-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCCCTCCCCCACCGACCCCA-5’

9. 95ºC 30 Sec. 95ºC 30 Sec. 95ºC 30 Sec. 95ºC 30 Sec. 72ºC 30 Sec. 72ºC 30 Sec. 72ºC 30 Sec. 72ºC 30 Sec. 60ºC 30 Sec. 60ºC 30 Sec. 60ºC 30 Sec. 60ºC 30 Sec. “THERMOCYCLING” • Denaturing Step • Primer Annealing Step • Elongation Step 95ºC 30 Sec. 72ºC 30 Sec. Temperature 60ºC 30 Sec. Time

10. Most PCR applications use 30 cycles (230 = 1.07 billion), representing an amplification of about 1 billion fold.

11. PCR Primers Design 1) Requires knowledge of the target sequence (i.e. we can’t use PCR to amplify an unknown sequence) 2) Use can designate subsection of gene to amplify. 3) Primers must have similar “melting temperatures” (e.g. 60ºC)

12. Calculating Melting Temperature (Tm) Based on thermodynamic binding properties of double-stranded DNA. G-C pairing has higher energy than A-T binding! There are many methods for calculating Tm, and generally speaking it is more important to use the same method (consistency) rather than making exact predictions (accuracy). Common method; Every G or C is assigned a value of 4ºC, and every A or T is assigned a value of 2ºC. Tm = 4(G + C) + 2(A + T) ºC

13. = 11 = 9 Tm = 4(11) + 2(9) Tm = 62ºC Calculating Melting Temperature (Tm) Tm = 4(G + C) + 2(A + T) ºC 5’-ACGTGTGTCAGCTGTAGTCG-3’ =4 x C =7 x G =3 x A =6 x T This method of calculating Tm is limited to short sequences, and really only useful in PCR primer design, where the annealing temperature is expected to be near 60ºC.

14. Calculating Melting Temperature (Tm) For longer DNA-DNA melting temperature calculations, the following formula is suggested; Tm = 81.5 + 16.6log(M of NaCl) + 0.41(%GC) – 0.65(%Formamide) – (500/length) Amount of Formamide in the reaction buffer Amount of Salt (NaCl) in the reaction buffer Length the DNA hybridization Percent of GC in the DNA hybridization This method is commonly used in calculating the Tm of oligonucleotide probes used in the DNA microarray assay platform (discussed later).

15. PCR Primers Design • Designate subsection of gene to amplify. • OR – • Designate the desired length of the PCR product (amplified DNA).

16. 2) Note that the complementary strand is present (but not shown in sequence file): GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCC 3) Primers are extracted from sequence: 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG-3’ 3’-CCTCCGTTGGCAATAGGTGGAGT-5’ 5’-ATGGAACACTGGGGGGAGCC-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCC-5’ Example of PCR Primer Design 1) Target Sequence: GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG

17. 5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG-3’5’-GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGG-3’ 3’-CCTCCGTTGGCAATAGGTGGAGT-5’ 5’-ATGGAACACTGGGGGGAGCC-3’ 3’-CCTACCTTGTGACCCCCCTCGGCTATGGGTCCTGTCCCGTCAGGACCTCCGTTGGCAATAGGTGGAGTCC-5’ Amplified Region, which includes Primer Sequences Primers (WRITTEN 5’ to 3’ !!!) “Forward” Primer: ATGGAACACTGGGGGGAGCC “Reverse” Primer: TGAGGTGGATAACGGTTGCCTCC

18. Computationally, PCR Primer design involves the coordination of; • Selecting 2 different primers that are: • Any distance apart (there are length limits in PCR!) • -OR- A specified distance apart (e.g. “PCR product will be 300 base-pairs long”) • -OR- Span a specific subsequence or “motif” • (e.g. “PCR product includes exons #1 and #2”) 2) Selecting 2 different primers that are nearly/exactly the same melting temperature (Tm). This is usually 60ºC. 3) Many times PCR Primers are designed with a “GC Clamp”. This simply means that the 3’ end of the primers is a G or a C, which binds more tightly (than AT) and the Polymerase initiates synthesis more efficiently. 4) Finally, better results from PCR reactions occur if the two primers are not complementary to each other, or contain extensive palindrome sequence.

19. x x x x x x • Forward Primer: GAGCCGATACCCAGGACAGG (18 bases, Tm = 60) • Reverse Primer: CCATGTGGCTGTCCCCAGCC (18 bases, Tm = 60) x x PCR Primer Design Demonstration: >unknown gene provided by Dr. Kane GGATGGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAACCGTTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTGGCTGGGGCCAGCCTCATGGAGGTGGCTGGGGCCAACCTCATGGAGGTGGCTGGGGTCAGCTCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGCTGGGGTCAAGGTGGTACCCACGGTCAATGGAACAAACCCAGTAAGCCAAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGGTAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTTATACATTTTGGCAGTGACTAT 70 Nucleotides Wide Primer Specifications: 60ºC, product bigger that 100 bp, GC clamps. • Forward Primer: GAGCCGATACCCAGGACAGG (20 bases, Tm = 66) • Reverse Primer: CCATGTGGCTGTCCCCAGCC (20 bases, Tm = 68)

20. PCR Primer Design Process. • Establish Specifications • Target Gene • PCR Product Size (not needed) • Tm of Primer pair (must be nearly/exactly the same) • GC Clamp (not needed) 2) Identify Candidate Primers START WITH 20 BASE PRIMER LENGTH!!! a) “Forward” Primer is derived from Target Sequence (5’-3’) b) “Reverse” Primer is; i) To the “right” or “3’ direction” of the Forward Primer ii) It is Complementary to the Target Sequence (3’-5’)!!! iii) It must be written 5’-3’ (inverted) to be correct convention. 3) Calculate Tm for each Primer a) If calculated Tm is NOT at specified temperature, ADD or DELETE bases from end. * OR * b) If calculated Tm is NOT at specified temperature, MOVE Primer sequence 1 or more bases. 4) Check Primers for “Primer-pair binding” (i.e. if the are primers significantly complementary with each other, they will bind to each other and decrease PCR reaction performance).