Molecular Bio. Exam 3, PCR and DNA Sequencing

who first proposed the theory of PCR

• early 1970s by G. Khorana et al

• his method was virtually forgotten, and then independently conceived some 15 years later by Kary Mullis

Why wasn’t PCR not practicable at first?

• thermostable polymerase

• oligonucleotide synthesis (primers)

Kary Mullis (1983)

created PCR, a technique for making copies, or amplifying, a specific sequence of DNA in a short period of time

process of PCR (as proposed by Kary Mullis)

• target DNA to be amplified is added to a tube, mixed with nuclotides (dATP, dCTP, dGTP, dTTP), buffer, and DNA polymerase

• paried set of forward and reverse primers are added — short single-stranded DNA oligonucleotides (20-30 bp long)

  • primers are complementary to nucleotides flanking opposite ends of target DNA

• reaction tube is placed in an instrument called a thermocycler (which will take DNA through a series of reactions called a PCR Cycle → denaturation, annealing, extension) (the cycle is repeated 20-30 times)

PCR Cycle

• Denaturation - heat to 94 C to 96 C

  • denatures and breaks up DNA strands

• Annealing (hybridization) - primers H-bond with complementary bases at the opposite ends of target sequence at 55 C to 65 C

  • cooling allows primers to bind (hybridize)

• Extension (elongation) - DNA Pol copies target DNA at 70 C to 75 C

  • DNA pol extends 3’ end of each primer

• at the end of one cycle, the amount of DNA has doubled & cycles are repeated 20-30 times

Advantages of PCR

• amplification of millions of copies of target DNA from a small amount of starting material in a short period of time

• use 2ⁿ to calculate the number of copies of DNA starting with 1 molecule of DNA (N represents the number of PCR cycles)

Applications of PCR

• making DNA probes

• studying gene expression

• detection of viral and bacterial infections

• diagnosis of genetic conditions

• detection of trace amount of DNA tissues found at crime scenes

Taq DNA polymerase

DNA pol isolated from a species known as Thermus aquaticus that thrives in hot springs

Why is the type of DNA pol used important?

dictates how well, how fast, and how accurately your DNA target is copied through repeated heating and cooling cycles. Different polymerases possess unique functional traits

Cloning PCR Products

a rapid and effective way to clone regions/ genes of interest (compared to using restriction digestion of DNA samples)

Disadvantage of PCR

• need to know something about the DNA sequence that flanks the gene of interest to design primers

• Taq polymerase puts a single adenine nucleotide on a 3’ end of all PCR products

  • this can be used to the researchers advantage: a T vector that has a single stranded thymine on each end so it can base pair with adenine in the PCR products

essential components of PCR

• thermostable DNA polymerase to catalyze template-dependent synthesis of DNA

  • for most applications: Taq pol remains remains the enzyme of choice

    • others may be used based on fidelity and ability to synthesize large products

• DNA primers

• dNTPs - building blocks for new DNA synthesis

• divalent cations - Mg2+ is an essential cofactor for thermostable DNA polymerases

• buffer to maintain pH

• monovalent cations

• template DNA

DNA primers in PCR

• pair of synthetic oligonucleotides to prime DNA synthesis (forward and reverse)

• the most critical aspect of PCR that influences efficiency and specificity of the amplification

• guidelines for design are largely qualitative and based on common sense instead of well-understood thermodynamics or structural principals

  • compliance with empirical rules does not guarantee success

deoxyribonucleotides (dNTPs) in PCR

• building blocks of new DNA synthesis

• standard reactions contains equimolar amounts of dATP, dCTP, dGTP, and dTTP

• too high concentration of dNTPs may be inhibitory to the reaction

  • if dNTP concentration is too higher, Mg2+ depletion occurs

  • each dNTP should be present at no more than 200 um (0.2 mM) in the final reaction concentration

Divalent Cations in PCR

• Mg2+ is an essential cofactor for thermostable DNA pol

• Mg2+ supports DNA synthesis and interacts with dNTPs during nucleotide incorporation

• optimal Mg2+ concentration depends on the reaction components, especially dNTP concentration, template, and primers

• typical starting concentration is 1.5-2.5 mM Mg2+; optimization may be needed (limits to MgCl makes the reaction more specific)

• too little Mg2+ reduces amplification; too much can result in an increase in nonspecific products

buffer in PCR

• maintains pH

• Tris-Cl, adjusted to pH between 8.3-8.8 at room temp, is the included standard concentration in PCRs at 10mM

• when incubated at 72 C, the pH of the reaction mixture drops to that of ~7.2, which is optimal for Taq pol

Monovalent cations

KCl at 50 mM is standard and works well for amplification of segments >500 bp; raising the concentration to 70-100 mM often improves the yield of shorter amplicons

template DNA in PCR

• closed circular DNA templates amplify less efficiently than linear DNA

• amplification of high-molecular weight DNA (>10kb) can be improved by digesting the template with RE that does not cleave within the targeted amplicon (before denaturing; dont want RE to digest any regions of interest)

• in theory PCR requires only a single copy of a target sequence for amplification

  • mammalian genomic DNA is often supplied up to 1 ug of DNA per reaction (50 ul). This would be 3×10^5 copies of single autosomal gene

  • yeast, bacterial, and plasmid DNAs the amount used is 10 ng, 1 ng, and 1 pg respectively

denaturation

• double stranded DNA denatures at temperatures that are determined in part by GC content

• higher GC = higher temp needed to separate strands

• longer DNA molecule = longer time needed to separate two strands

• usually, 94-95 C is used (which is the highest temp. that the enzyme can endure for 30+ cycles)

  • in the first step, sometimes a longer denature time is allowed (>5 minutes)

Annealing

• critical step for optimizing reaction (in this step, if correct temp was used and if the correct amount of MgCl2 was used)

• usually set to temp 3-5 below the calculated Tm (the temp when primers dissociate from the template) (temp when the primers dissociate from template strand)

• this is a step when you need to optimize your experiment by progessively lowering the annealing temp in a series of reactions

• higher temp = more specific

extension of primers

• carried out at the optimal temp for the thermostable DNA pol

• usually Taq pol, which is 72 C

• polymerization rate for taq pol is ~2000 nucleotides / min

  • by convention, extension is carried out for 1 min for every 1000 bp of product

  • for the last cycle, often use time that is three time longer than those of previous cycles to allow for compensation of all amplified targets

potential PCR inhibitors

• proteinase K (often present in extraction kits for DNA) (breaks down proteins, so if a solution will breakdown Taq pol)

• phenol

• centrimonium bromide (CTAB) (disrupts cell wall)

• EDTA (kelating agent that binds and remove Mg from solution)

• hemoglobin

• gel loading dye (bromophenol blue)

• alcohol / ethanol

• inhibitors often are present in the prepared template DNA

• many problems with PCR can be cured by simply cleaning up template appropriatly (ethanol precip. or chloroform extraction)