DNA synthesis in the lab

• Main ingredients for PCR:

  • Template
  • DNA polymerase
  • Forward and reverse primers
  • dNTPs
  • Buffer
• Buffers have the optimal [Mg2+], pH and ionic strength
• During denaturation, the mixture is heated to separate DNA strands
• DNA strands are held together by weak H bonds, so they can be separated by heating
• With increasing temperatures, DNA strands will become separated so the absorbance at 260nm will increase
• During annealing, the mixture is cooled to allow specific primers to H bond to the region of interest
• We can design primers so that they only amplify what we are interested in by binding the relevant region
• Unlike replication, we often don't want to copy the entirety of the DNA in PCR
• Reverse primers are also added so that replication occurs in the desired 5' to 3' direction for the complementary strand
• Appropriate melting temperatures are required for primers
• Primers must not anneal to themselves or each other
• Mg2+ is required for the functions of DNA polymerase
• adding too much Mg2+ will shield the negative phosphates of DNA, promoting base pairing
• increased base pairing increases the melting point of the DNA and reduces the specificity of primer binding
• Complementary bases undergo H bonding
• Polymerase catalyses the formation of phosphodiester bonds, adding nucleotides to the growing chain
• The amplicon becomes the template for the next cycle, leading to doubling each cycle
• Amplicon = newly synthesised DNA
• Taq polymerase is a heat-resistant polymerase enzyme
• DNA has a constant charge to mass ratio due to the negative phosphates of the DNA backbone
• Small DNA strands have less charge, so will therefore migrate less
• Spectrophotometry cannot be used to determine the size of DNA as it only measures the bases
• Dyes are added to see DNA or RNA
• Dyes can intercalate with DNA and fluoresce under UV light
• The length of variable number tandem repeats will vary between individuals
• Size of amplified DNA will increase with the number of repeats, which will manifest as higher bands
• Reverse transcriptase is a DNA polymerase that uses an RNA template to make cDNA
• Viruses use reverse transcriptase to produce DNA copies of their RNA genome
• Reverse transcriptase requires primers
• Ribonucleases are found on skin, in water, etc.
• RNA contaminated by ribonucleases will be degraded
• Reverse Transcriptase PCR is commonly used to study mRNA expression
• Reverse transcriptase PCR

  1. Isolate RNA
  2. Make cDNA
  3. PCR of gene of interest
  4. Analysis
• Sanger Sequencing uses a mixture of deoxy analogs of dNTPs in PCR
• Limitations of Sanger Sequencing

  • Cannot amplify for lots of base pairs
  • Requires information about the sequence to design primers
•  Next-Generation Sequencing

  • Sequences many different strands at one time
  • Does not require information about the sequence
• How does Next-generation sequencing work?

  PPi released from the addition of dNTPs is detected