Lab 8

• Polymerase Chain Reaction (PCR)

  The polymerase chain reaction (PCR) is a biochemical technology in molecular biology, to amplify a single or a few copies of a piece of DNA (Target), generating thousands to millions of copies of this particular DNA sequence

  
• Purpose of PCR

  To measure the amount of nucleic acid in a sample and as such estimate the expression of certain genes

  
• PCR components


  • DNA template (target) to be amplified
  • Two primers
  • Taq polymerase (DNA polymerase)
  • Deoxynucleotide triphosphates (dNTPs)
  • Buffer solution containing Divalent cations (i.e: Mg2+) required as a co-factor for thermostable DNA polymerase

  
• Taq polymerase


  DNA polymerase from the hyperthermophilic organisms Thermus aquaticus, commonly used because it is thermally tolerant (survives the melting T of DNA denaturation) → the process is more specific since higher temperatures result in less mismatch and more specific replication
• Reverse Transcription Polymerase Chain Reaction (RT-PCR)


  RT-PCR is used to convert RNA into complementary DNA (cDNA) and then amplify this cDNA for analysis. It is a variation of the standard PCR (Polymerase Chain Reaction) technique that is primarily used to amplify and analyze DNA.

  
• Steps of RT-PCR


  1. Reverse Transcription: RNA is converted to cDNA using reverse transcriptase

  2. Amplification: cDNA is amplified using PCR technique

  3. Detection

  
• One-Step RT-PCR


  cDNA synthesis and PCR amplification are coupled

  
• Two-Step RT-PCR


  cDNA synthesis and PCR amplification are uncoupled

  
• Quantitative Polymerase Chain Reaction (qPCR)

  Using fluorescent dyes such as SYBR Green or fluorescent-containing DNA probes such as TaqMan, we can measure the amount of amplified product as the amplification progresses. Special thermal cyclers are used to monitor the amount of product during the amplification.

  
• SYBR Green method


  Intercalating dye that binds to dsDNA
• TaqMan probe method


  Specific probe that binds to a sequence of interest. Upon synthesis of complementary DNA strand, the probe is cleaved separating the reporter from the quencher, resulting in a fluorescent signal.

  
• Cycle threshold (Ct)


  The number of cycles required for the fluorescent signal to cross the threshold (ie exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (ie the lower the Ct level the greater the amount of target nucleic acid in the sample).

  
• Housekeeping genes in qPCR

  Reference genes or House Keeping genes, such as actin, are used in qPCR to account for variations in the starting material, reverse transcription efficiency, and qPCR efficiency, to ensure that any changes in the quantification of the gene of interest are due to biological differences and not technical variations.

  
• Applications of PCRs


  • DNA amplification
  • Gene expression analysis: to measure the amount of mRNA present for a particular gene and infer its expression level using qPCR
  • Genetic testing: Diagnosis of genetic diseases, identification of genetic variations (single nucleotide polymorphism) and forensic DNA analysis
  • DNA sequencing: used in conjunction with DNA sequencing techniques to amplify specific regions of DNA before sequencing
  • Evolutionary and population studies: analyze genetic variation within and between populations
  • Infectious disease diagnosis: detection of infectious agents such as viruses, bacteria, and parasites
  • Environmental and food testing: to detect and quantify microorganisms or pathogens present in environmental samples or food products
  • Mutation detection: used to detect mutations or genetic abnormalities associated with various diseases, including cancer
  • DNA cloning: used to generate DNA fragments that can be cloned into vectors for various molecular biology applications

  
• Case 1: Detection of infectious pathogens using PCR

  • Diagnosis of a viral respiratory infection in a patient presenting with fever, cough, and respiratory distress. Respiratory samples (nasopharyngeal swab or sputum) are collected from the patient for analysis. Nucleic acid extraction is performed to isolate the viral RNA from the respiratory samples. Polymerase chain reaction (PCR) is performed to amplify specific regions of the viral genome using primers designed to target conserved regions of the virus. If the viral nucleic acid was successfully amplified, the patient is diagnosed with viral respiratory infection.
• Case 2: Early Detection of Breast Cancer using PCR

  • Fine needle aspiration (FNA) biopsy of suspicious breast lesion previously identified on imaging. Polymerase chain reaction (PCR) is performed targeting specific biomarkers associated with breast cancer such as HER2/neu, estrogen and progesterone receptors. Amplification of the HER2/neu gene and overexpression of estrogen and progesterone receptors indicates the patient is diagnosed with hormone dependent breast cancer, which is useful information for treatment decision.