Lec3-PCR & Electrophoresis 2024 PDF

 ผลการเรียนรู้ท่ีคาดหวัง(Learning Outcomes) เมือ่ สิน้ สุดการสอนรายวิชานีน้ สิ ติ สามารถ 1. อธิบายความสาคัญของส่วนประกอบทีใ่ ช้ในการทา PCR และปัจจัยทีเ่ กีย่ วข้องใน ปฏิกริ ยิ า PCR ได้ 2. คานวณสารละลายและเตรียมสารละลายองค์ประกอบ PCR ได้อย่างถูกวิธี 3. ประยุกต์ใช้เทคนิค electrophoresis และเต...

 ผลการเรียนรู้ท่ีคาดหวัง(Learning Outcomes) เมือ่ สิน้ สุดการสอนรายวิชานีน้ สิ ติ สามารถ 1. อธิบายความสาคัญของส่วนประกอบทีใ่ ช้ในการทา PCR และปัจจัยทีเ่ กีย่ วข้องใน ปฏิกริ ยิ า PCR ได้ 2. คานวณสารละลายและเตรียมสารละลายองค์ประกอบ PCR ได้อย่างถูกวิธี 3. ประยุกต์ใช้เทคนิค electrophoresis และเตรียม gel เพือ่ ใช้ตรวจสอบผลิตภัณฑ์ จากปฏิกริ ยิ า PCR ได้อย่างถูกวิธี 4. ตรวจสอบ อ่านผลและวิเคราะห์ผลิตภัณฑ์จากปฏิกริ ยิ า PCR ด้วยเทคนิค electrophoresis ได้ Detection of Leishmania and Trypanosoma DNA in Field-Caught Sand Flies from Endemic Areas of Leishmaniasis in Southern Thailand. ITS1 region PCR investigation Generation of PCR Setting up PCR assay ❑ PCR Reagents ▪ PCR Reagents ▪ DNA Polymerases ▪ DNA Template ❑ PCR Assay Design and Optimization ▪ PCR condition ▪ Primer Design for a PCR Assay ▪ Gradient Optimization for PCR Assays ❑ PCR Instrumentation ▪ Thermal cycler ❑ PCR Analysis ▪ Gel Electrophoresis ▪ Semiquantitative Analysis  PCR Reagents ❑ PCR reaction : Conventional and Commercial ❑ PCR reaction components include ▪ Buffer has some MgCl2; Mg2+, Tris-HCl, Triton X-100, Tween ▪ Deoxyribonucleotides (dNTPs) ▪ Oligonucleotide primers ▪ DNA polymerase: Taq polymerase; Hot start ▪ Purified nucleic acid template  PCR Reagents purified nucleic acid ❑ Nucleic acid purification and quality assessment are important steps in the PCR experimental workflow. ❑ It is important to use proper techniques to isolate and purify nucleic acids because this step can affect amplification reactions.  Sample recipe & use of master mix ❑ A typical recipe for a PCR reaction would be something like this: ▪ 25-100ng/μl Template DNA ( ≤ 1 μg)——–––––-––--–––1 μl ▪ 10X PCR buffer (contains MgCl2; 0.5-2.5 mM)—---––2.5 μl ▪ 10 mM dNTPs (20-200 μM)————––––––––—---—–2.5 μl ▪ 10 μM Forward primer (0.1-0.5 μM) —–––--––-———0.5 μl ▪ 10 μM Reverse primer (0.1-0.5 μM)——–--–––-–——-0.5 μl ▪ 5U/μTaq polymerase (1-2.5 U)—––––––---–––-———0.5 μl ▪ ddH20———————–––––––––––––––––––-–——-17.5 μl ▪ Total volume: 25 μl  Primer Design for PCR Assay ❑ A successful PCR assay requires efficient and specific amplification of the product. ❑ Both the primers and the target sequence can affect the efficiency, specificity, and accuracy of PCR assays. ❑ Therefore, care must be taken when choosing a target sequence and designing primers. ❑ The use of PCR primers specifically designed and validated for PCR assays with target of interest is highly recommended.  Primer Design for PCR Assay ❑ When designing primers for a PCR assay, follow these steps: ▪ Check the literature and databases for existing primers. ▪ Choose a target sequence. ▪ Design primers. ▪ Check primer specificity. ▪ Assess primer properties (melting temperature [Tm], 2nd structure, complementarity). ▪ Determine PCR product properties ▪ Optimize the protocol.  Primer Design for PCR Assay ❑ Check the literature and databases for existing primers:  Primer Design for PCR Assay ❑ Design novel primers: ▪ Search for reference target sequences ▪ Design primer: program online – GenScript, Web primer, Primer 3 Plus, Primer-BLAST ▪ Checking specificity (BLAST)  Primer Design for PCR Assay ❑ Choosing a Target Sequence: ▪ Plan to amplify 75-200 bp product (at least 75 bp to easily distinguish it from any primer-dimers ). ▪ Avoid regions that have secondary structure. ▪ Avoid regions with long (>4) repeats of single bases. ▪ Choose a region that has a GC content of 50–60%. Search for reference target sequences  Primer Design for PCR Assay ❑ Choosing a Target Sequence:  Primer Design for PCR Assay ❑ Designing Primers: ▪ Design primers that have a GC content of 50–60% ▪ Strive for a Tm between 50 and 65°C. ▪ Avoid 2nd structure; adjust primer locations so that they are located outside 2nd structure in the target sequence. ▪ Avoid repeats of Gs or Cs longer than 3 bases. ▪ Place Gs and Cs on ends of primers. ▪ Check the sequence of forward and reverse primers to ensure no 3' complementarity (avoid primer-dimer formation). ▪ Verify specificity using tools such as the Basic Local Alignment Search Tool BLAST or Primer-BLAST. Design primer: program online –Primer-BLAST  Primer Design for PCR Assay ❑ Choosing a Target Sequence: ❑ Position of primer for Leishmania DNA: ▪ Forward primer; LeF : 5′-TCC-GCC-CGA-AAG-TTC-ACC-GAT-A-3′ ▪ Reverse primer; LeR : 5′-CCA-AGT-CAT-CCA-TCG-CGA-CAC-G-3′ ❑ PCR product = 379 bp  Primer Design for PCR Assay ❑ Verify specificity using tools such as Primer-BLAST  PCR Condition ❑ Cycle number and length. ▪ In general, the cycle number should be kept to the minimal number needed to generate sufficient product required for further analysis. Initial denaturation Denature Annealing Extension Final extension Hold  Gradient Optimization for PCR ❑ Optimizing the annealing temperature of PCR assay is one of the most critical parameters for reaction specificity. ▪ Too low → amplification of nonspecific PCR products. ▪ Too high → reduce the yield of a desired PCR product. ❑ Even after calculating the Tm of a primer, may need to determine the annealing temperature empirically. ❑ This involves repeating a reaction at many different temperatures. ❑ Similar time-consuming tests may also be required to optimize the denaturation temperature.  Gradient Optimization for PCR ❑ To find the optimal annealing temperature for reaction, test a range of temperatures above and below the calculated Tm of the primers. ❑ Analyze the results using agarose gel electrophoresis. ❑ If nonspecific amplification has occurred, additional bands will appear on the gel. ❑ The optimal annealing temperature is the one that results in the highest yield with no nonspecific amplification. ❑ If satisfactory results are not obtained at any annealing temperature, additional optimization steps must be taken, and primer redesign may be necessary. Gradient Optimization for PCR ❑ Gradient optimization of a PCR assay. ▪ All reactions were evaluated in a single run. ▪ Four different primer sets (A, B, C, and D) were designed and tested for amplification. ▪ Arrows indicate the annealing temperature that provided the highest specificity while maintaining good yield. ▪ Yellow box indicates optimal temperatures. ▪ M, markers; Tm, melting temperature. Gradient Optimization for PCR  PCR Analysis ❑ Depending on the information desired. ▪ Gel Electrophoresis ▪ Denaturing gradient gel electrophoresis (DGGE) ▪ Temporal temperature gel electrophoresis (TTGE) ▪ Semiquantitative Analysis ▪ etc.  Gel Electrophoresis ❑ Is a common technique to detect the presence or absence of the target sequence and the length of the fragment. ❑ The results can be visualized by ethidium bromide or non-toxic dyes such as SYBR® green. ❑ The intensity of the band can be used to estimate the amount of product of given molecular weight relative to a ladder. ❑ Gel electrophoresis also shows the specificity of the reaction, where the presence of multiple bands indicates secondary amplification products.  Gel Electrophoresis ❑ Agarose is a common material for gel electrophoresis. ❑ When agarose solution is heated, it is in liquid form and as it cools the matrix gets formed. ❑ As we increase the concentration of agarose (g/ml) the pore gets smaller..  Gel Electrophoresis ❑ Agarose gel concentration for resolving linear DNA molecules. Agarose DNA size range Low concentration gel separates conc. (%) (bp) large DNA molecules more clearly. 0.2 5000-40000 0.4 5000-30000 0.6 3000-10000 Intermediate concentration gel 0.8 1000-7000 separates DNA molecules almost uniformly. 1 500-5000 1.5 300-3000 2 200-1500 3 100-1000 High concentration gel separates small DNA molecules more clearly.  Gel Electrophoresis ❑ Staining of DNA in gel : ❑ Agarose gel ▪ Ethidium bromide ▪ GelRed ▪ RedSafe ▪ SYBR green ▪ Novel Juice ❑ Polyacrylamide gel ▪ Silver staining ▪ FAM, NED, VIC.  Gel Electrophoresis ❑ DNA (band) size estimation: ▪ Use the relative mobility (Rf ) of the DNA molecule in the gel (Distance travel vs. size) ▪ Compare your DNA sample to a standard of known sizes (DNA ladder)  PCR Troubleshooting ❑ No Band or Faint Band ❑ Causes Related to Cycling Times and Temperatures ▪ Too few cycles were used ▪ Extension time/Annealing time was too short ▪ Annealing temperature was too high ▪ Denaturation temperature was too low ▪ Denaturation time was too long/ too short ❑ Causes Related to PCR Components ▪ dNTP concentration was too high/ too low ▪ PCR product has high GC content (>65%) ▪ Template was damaged or degraded or not enough or contained inhibitors ▪ Primers/water/dNTPs contained impurities ▪ Not enough Mg2+ ▪ Primer concentration was too high/ too low  PCR Troubleshooting ❑ Nonspecific Bands or Primer-Dimers ❑ Causes Related to Cycling Times and Temperatures ▪ Too many cycles were used ▪ Extension time/Annealing time was too long ▪ Annealing temperature was too low ▪ Thermal cycler ramping speed is too slow ▪ Calculated primer Tm was inaccurate ❑ Causes Related to PCR Components ▪ Primers contain impurities ▪ Too much primer was added ▪ Primers were designed or synthesized incorrectly by user or manufacturer ▪ Impure dNTPs were used ▪ Too much Mg2+ was added ▪ Impure water was used  PCR Troubleshooting ❑ Smeared Bands ❑ Causes Related to Cycling Times and Temperatures ▪ Too many cycles were used ▪ Extension time/Annealing time was too long ▪ Annealing temperature was too low ▪ Thermal cycler ramping speed is too slow ▪ Calculated primer Tm was inaccurate ❑ Causes Related to PCR Components ▪ Too much primer was added ▪ Template contained an exonuclease or was degraded ▪ Primers contained impurities ▪ Primers were designed or synthesized incorrectly by user or manufacturer ▪ Impure dNTPs/water were used