Understanding the process of PCR

Understanding PCR

• PCR, or polymerase chain reaction is a widely used technique for creating multiple copies of a DNA template
• Step 1, is the denaturing step-here a doubled stranded DNA template is denatured into two single strands by heating the reaction mixture to 95°C
• Step 2, known as the annealing step is where forward and reverse primers attach themselves to single-stranded DNA templates, usually around 55°C
• Primers' exact annealing temperature depend on primer specifics
• Step 3, is the elongation step where Taq or Pfu DNA polymerase binds to the 3' end of the primer
• Taq and Pfu polymerase are thermophilic or heat-loving polymerases
• Nucleotides (dATP, dCTP, dGTP, dTTP) are added to synthesise a new DNA strand at 72°C
• These three steps are repeated around 30-40 times in order to amplify the target DNA sequence
• A typical polymerase chain reaction contains the following in a 50 µl reaction in a 0.2 ml tube placed in a PCR machine
  • 10X Pfu DNA Polymerase Buffer with MgSO4 at 5 μι
  • dNTP mix at 10mM each in stock at 1 μι
  • Forward primer at 10 µM stock at 1 μι
  • Reverse primer at 10 µM stock at 1 μι
  • DNA template at 1 ng/µl at 5 μι
  • Pfu DNA Polymerase at 1u/µl at 1 μι
  • Sterile Water at 36 μι
• The final concentration of the 10x Pfu DNA Polymerase Buffer with MgSO4 in the 50 µl PCR reaction is 1x
  • Calculation includes 5 µl of 10x Pfu DNA Polymerase Buffer with MgSO4 added to a 50 µl total volume
• The final concentration of the dNTP mix in the 50 µl PCR reaction is 0.2mM (or 200 μΜ)
  • Calculation included 1 µl of dNTP mix (10mM each in stock) added to a 50 µl total volume
• The final concentration of the forward primer in the 50 µl PCR reaction is 0.2 μΜ or (200 nM)
  • Calculation included 1 µl of forward primer (10 µM stock) added to a 50 µl total volume
• There is 5 ng of template DNA is in the 50 µl reaction, calculated below
  • 5 µl of the DNA template (1 ng/µl) was added to the reaction mixture
  • 1 ng/µl times 5 µl equals 5 ng
• The PCR reaction contains Pfu DNA polymerase as it is involved in DNA synthesis
  • It is a thermophilic or heat-loving polymerase and retains its activity even after it has been heated to 95°C multiple times
  • Normal DNA polymerases would be permanently inactivated at temperatures >65°C
  • Active at 72°C and keeps primers binding non-specifically to template DNA

DNA Sequencing

• DNA sequencing uses a chain termination method(also referred to as Sanger sequencing)
• A traditional DNA sequencing reaction mixture includes:
  • A DNA Template and primer sequence
  • dNTPs like dATP, dCTP, dGTP and dTTP
  • A DNA Polymerase
  • Low levels of ddATP (or ddCTP or ddGTP or ddTTP)
• A classical Sanger sequencing reaction labels one of the dNTPs with 35S
• Making the single strand of DNA that is produced radioactive and able to be detected by X-ray film
• In automated chain-termination DNA sequencing, the DNA sequencing reaction mixture still contains
  • a DNA template and primer sequence
  • dNTPs (dATP, dCTP, dGTP and dTTP)
  • a DNA polymerase
  • low levels of ddATP, ddCTP, ddGTP, ddTTP (all four)
• Each dideoxy nucleotide is labelled with a different florescent dye
• The denatured reaction mixture is separated by capillary electrophoresis
• Automation typically gives around 1000 bases of good quality DNA sequence data from a single primer/DNA template reaction
• Most university researchers send DNA samples to commercial companies for a cost of approximately £3.00 per reaction

DNA and cDNA Libraries

• The (haploid) human genome consists of approximately 3 x 10º base pairs
• With the recognition sequence GATC, the number of fragments approximately will be 11,718,750 or 1.171875 x 107
  • The sequence GATC will occur by chance approx. every 256 base pairs
  • Calculation is 3 x 109/256 = 1.17 x 107 fragments
• With the recognition sequence GGATCC, the number of fragments approximately will be 732,421.875 or 7.33421875 x 105
  • Cuts every 46 base pairs equals 4096 bp
• A genomic library is a collection of cloned DNA fragments into a vector, every genomic DNA sequence from an organism represented at least once
• A cDNA library is a collection of complementary DNA (cDNA) clones produced from the entire mRNA population isolated from tissue/organ cells
• Genomic libraries contain the same DNA sequences as they are made from genomic DNA
  • This is because in almost every cell of the body, genomic DNA which is identical
• cDNA library will only represent the genes expressed in the tissue that the mRNA was isolated from
  • Some genes are expressed in all types of cells (housekeeping genes)
  • Some genes are expressed in a select number of tissues (but not all tissues)
  • Some genes will only be expressed in that tissue, called 'tissue specific'
• A cDNA library prepared from pancreatic ẞ cells would be the most appropriate starting point for isolating proinsulin cDNA
• As insulin is produced in the pancreas

Analysis of phGH

• Cloned human growth hormone, produced by bacteria, can treat short stature individuals
• The human growth hormone gene encodes mRNA, which is 833 bases in length, the entire gene is 47,000 base pairs long
• The cloning procedure used to produce growth hormone in E. coli copies the growth hormone mRNA to produce cDNA
• mRNA is used as starting material in eukaryotes, rather than genomic DNA, reasons below:
  • mRNA contains no introns and a single long open reading frame for protein
  • Making it easy to handle by gene cloning techniques
  • Genomic DNA can contain exons and introns (so can be 10-200 kb long)
  • Genomic also has promoter/regulatory sequences alongside transcription and translation termination sequences
  • making the use of numerous introns a usual cause for a great length
• Reverse transcriptase is the enzyme that can copy cDNA from RNA
  • It comes from retroviruses AMV and MMLV
  • Retroviruses have ssRNA genome that replicate via dsDNA intermediate
• A plasmid called phGH was constructed to express the human growth hormone in E. coli
  • the open reading frame (ORF) from the human growth hormone cDNA was cloned in front of the lac promoter/operator region
• The plasmid phGH ampicillin resistance gene, an antibiotic-resistance gene allows the plasmid to grow allowing E. coli to grow in the presence of ampicillin
  • Ampicillin acts as an irreversible inhibitor of cell wall creating enzyme transpeptidase, which needed by bacteria, leading to cell lysis
  • The ampicillin resistance gene (amp) produces beta-lactamase to degrade ampicillin
• ori meaning origin of replication, All plasmids contain their own that are separate from origins found on the bacterial chromosome
  • Which means that plasmids replicate themselves within the cell independently
  • The present type of ori in a plasmid determines the plasmid copy number within a cell
  • The plasmid copy number for ori from pBR322 is between 30-40 plasmids per cell, and for pUC18 it 500 plasmids
• The longest open reading frame in the bottom reading frame corresponds to the known amino acid sequence of the human growth hormone shown
• With BamHI, one DNA fragment would be produced with this restriction enzyme as it cuts the plasmid once
• Cutting with enzymes such as these linearises the plasmid leading to a 4,000 bp total length
• With EcoRI, two DNA fragments would be produced
• With BamHI and EcoRI, three DNA fragments would be produced