MLAB 2378 Fundamentals of Molecular Diagnostics

Overview of Unit 10 Nucleic Acid Amplification

This chapter goes from very basic to pretty detailed. We want to learn the basics. What reacts with what to give a positive reaction. Whether it’s PCR or the PCR applications, I want you to focus on the basic principle. The following notes are to help you focus your studies.

USE THE PICTURES in the book. If you do not refer to the diagrams illustrating what the words in the chapter are explaining you will get lost.

  1. Kary Mullis conceived the idea of DNA amplification in-vitro in 1983.
  2. Each PCR amplification cycle consists of three steps: denaturation, primer annealing and extension.
  3. The components necessary for PCR include:
  4. Template to be copied
  5. Primers
  6. Nucleotides
  7. Polymerase buffer
  8. Polymerase enzyme
  9. Each step has an optimal temperature for the appropriate reaction to occur. This usually requires 3 temperatures: denaturation, annealing and extension.
  10. If the annealing and extension temperatures are close enough the reaction can proceed with only 2 steps.
  11. The annealing temperature is determined by calculating the temperature of melting.
  12. Primers are analogous to probes used in the blotting and hybridization procedures.
  13. Two primers are necessary, a forward and reverse. When the double stranded DNA denatures into two strands the order of the nucleotides for each strand is anti-complementary and one will be the 5’-3’strand, the other the 3’-5’ strand. For amplification to occur a primer must be developed for each strand.
  14. Developing the primers is dependent upon knowing the sequence of the target DNA to be analyzed so that it is complementary to the target.
  15. The %GC and length of the primer affect the optimal conditions at which the primer will bind to target.
  16. Both primers should have the similar temperatures of melting (Tm) so the annealing temperature will be the same. Remember, denaturing (breaking apart) the double stranded DNA results in 2 strands with different nucleotide sequences which are anti-complementary.
  17. Tm can be adjusted by:
  18. Increasing the length of primers
  19. Placing primers in areas with more or fewer Gs and Cs
  20. Mispriming can occur.
  21. Fragment synthesized from mispriming will consume components needed to amplify true target.
  22. Will interfere with proper interpretation or sequencing.
  23. Primer-dimer occurs when primers bind to each other instead of the target.
  24. The entire primer does not have to bind to template however 3’ nucleotide is critical for extension to occur.
  25. DNA template may be single or double stranded.
  26. For routine clinical analysis 100 ng to 1ug of DNA is used.
  27. The best DNA templates are in good condition, free of contaminating protein, and without nicks or breaks. Templates with high GC content and secondary structure may prove more difficult for amplification.
  28. Deoxyribonucleotide triphosphates (dNTPs) are the building blocks of DNA. You must add enough adenine, thymine, guanine and cytosine to the reaction mixture to support the amplification. If you run out of dNTPs then the amplification will stop. Remember you are duplicating the DNA strands which mean there must be sufficient quantities of bases in the reaction mixture to extend the target DNA strands.
  29. Taq polymerase is the DNA polymerase most frequently used. It can be added at the beginning of the procedure and maintains its activity throughout the healing and cooling cycles.
  30. Tth polymerase is more frequently used in reverse transcriptase (RT-PCR) where the starting material is RNA.
  31. PCR buffers provide the optimal conditions for enzyme activity.
  32. Potassium chloride, ammonium sulfate or other salts of monovalent cations are critically important. They affect the denaturing and annealing temperatures of the DNA and enzyme activity. Increase in salt concentrations slows down denaturation of long DNA products so short products will amplify preferentially.
  33. Magnesium chloride (MgCl2)affects primer annealing. Requirements will vary with each reaction, each NTP uses one magnesium atom. Also, EDTA or other chelators will lower magnesium available for the enzyme.e Too little magnesium results in lower PCR yield. High levels promote misincorporation and increase yield of non-specific products. DNA samples with EDTA must have Mg adjusted accordingly. Recommended rage of MgCl2 is 1-4nM in a standard reaction.
  34. Tris buffer important for optimal enzyme activity and accurate amplification: 10mM Tris-HCL maintains proper pH between 8-9.5.
  35. Accessory components contribute to stability of enzyme.
  36. Bovine serum albumin (BSA) binds inhibitors, stabilizes enzyme.
  37. Dithiothreitol (DTT) provides reducing conditions, enhances enzyme.
  38. Formamide lowers denaturing temperature of DNA with high secondary structure.
  39. Chaotropic agents reduce secondary structure.
  40. Master mix may be proprietary or “home brewed” contains buffer, other ingredients mixed with nucleotide bases and stored in aliquots. Enzyme, target and primers are then added. Some master mixes will include primers so only have to add target. Similar to buying a cake mix or mixing dry ingredients for a cake and storing it. Then only need to add your wet ingredients to make your cake, saves time.
  41. Thermal cyclers have vastly increased the efficiency for performing the PCR. Tubes with necessary “ingredients” are placed in the thermal cycler which automatically heats and cools the contents to the necessary temperatures
  42. Upon completion of PCR the product is analyzed using gel or capillary electrophoresis. Bands will form which will indicate the presence or absence of the product.
  43. Controls
  44. A ladder is run to verify size of the product.
  45. Positive control ensures that enzyme is active, buffer is optimal, primers are priming right sequence and thermal cycler is cycling appropriately.
  46. Contamination or reagent control has all components EXCEPT the target to detect that reaction mix was not contaminated with DNA or products from a previous run.
  47. Negative control has DNA which lacks the target and will detect primer annealing to unintended sequences of DNA.
  48. Amplification control has primers to a DNA target different than the one being tested for to ensure that all reagents are working properly even if the test sample was not amplified. Most important when PCR is reported out as “positive” or “negative”, negative meaning target not present.
  49. Controlling contamination is ESSENTIAL in PCR since, theoretically, a single DNA molecule will give rise to product. Contamination is controlled both PHYSICALLY and CHEMICALLY.
  50. Physical setting is critical; pre-PCR must be separate from post-PCR analysis. Separate rooms are BEST but can also utilize isolation cabinets. Equipment and all supplies MUST be dedicated to either pre- or post-PCR.
  51. Ultraviolet light catalyzes single- and double-strand breaks in the DNA. Isolation cabinets run UV light for 20 minutes after box is used. UV light more effective with by addition of psoralens to amplification products after analysis. Efficiency of UV light depends on wavelength, energy and distance of light source. Take care to avoid exposure to skin and eyes. Will also damage some plastics so take care not to leave in UV light.
  52. Solution of 10% bleach used to wipe down EVERYTHING. Have one tech perform procedure other tech note everything touched. It’s amazing what you will discover that you had not even thought of to decontaminate.
  53. Deoxy Uracil Triphospate- uracil-N-glycosylase (dUTP-UNG). Remember that uracil will bind to thymine. So, if dUTP is added instead of dTTP the dUTP will be incorporated instead of dTTP in the PCR product. The enzyme UNG will degrade any nucleic acid containing uracil, such as contaminating product from previous reactions. A short incubation with UNG is required prior to running the PCR. . Some polymerase enzymes are not as efficient in incorporating the dUTP so it may not work for some procedures. his system will not work with some types of PCR. It is used routinely in real-time PCR.
  54. Prevention of mispriming
  55. Misprimes can be prevented by good primer desing and optimal amplification conditions.
  56. To further prevent mispriming hot-start PCR can be used. This can be done in three ways:
  57. Reaction mixes prepared on ice and placed in thermal cycler that has been pre-warmed to denaturation temperature.
  58. Use wax barrier, bead of wax placed in reaction tubes with all components of the reaction mix except enzyme and template. Heat tube to 100C to melt wax, cool to RT, wax will float to top and congeal. Add template and enzyme to top.
  59. PCR product clean up involves removing extraneous products and/or residual components of the reaction mixture.
  60. PCR Modifications are done to detect multiple targets in the same run, using RNA as templates, using amplified products as templates or quantitating the starting template.
  61. Multiplex PCR– adding more than one primer pair to perform multiple amplifications at one time.
  62. Reverse-transcriptase PCR – Uses RNA as the starting material.
  63. Nested PCR – increases sensitivity and specificity. Uses two pairs of primers to amplify a single target in two SEPARATE PCR runs.
  64. Real-time (Quantitative ) PCR – uses a dye such as EtBr or SYBR green which fluoresces to monitor accumulation of PCR products during PCR in real time. Uses a probe attached to a fluorescent dye on one end and another dye or nonfluorescent molecule that pulls fluorescent energy from the 5’ dye (quencher) on the other.