These are comprehensive lecture notes for the BIOC0005 Week 5 to Week 6 lectures, which consist of 7 lectures in total. BIOC0005 is taken by 2nd year BSc students studying Molecular Biology, Biochemistry and other life sciences degrees. Important knowledge/exam points are highlighted or typed in re...
Polymerases make nucleic acid polymers (ie. DNA or RNA)
Di er in the product they make and what they use as template strand.
• DNA polymerase - DNA dependent DNA polymerase
• RNA polymerase - DNA dependent RNA polymerase
• Reverse transcriptase - RNA dependent DNA polymerase
• RNA replicase - RNA dependent RNA polymerase.
Some polymerase extend an existing strand instead of making a new strand
• Terminal deoxynucleotidyl transferase (TdT) - DNA dependent DNA polymerase
• polyA polymerase - RNA dependent RNA polymerase.
DNA polymerase
E coli DNA polymerase I.
• Has 3 separate activity domains:
◦ polymerase activity: synthesis
◦ proofreading activity: checks for correct bases and cuts o wrong ones
◦ exonuclease activity: removes primers on lagging strand.
• First two domains make up the Klenow fragment, third subunit can be cleaved o by proteases.
◦ Klenow fragment has many applications.
Thermostable DNA polymerases such as Taq polymerase is used in PCR.
• Original PCR used Klenow. But was not e cient because gets killed by heat. Replaced by thermal stable
polymerase.
• Taq polymerase: optimum activity is 75-80ºC.
◦ Proofreading domain is not functional. (Drawback - lower delity)
◦ Does not have exonuclease activity.
◦ Lack of proofreading often leads to addition of 3' overhangs of a single A to PCR products.
• DNA polymerase from hyperthermophilic bacteria and Archaea have proofreading activity.
• Pfu polymerase from archaean.
◦ More expensive than Taq but have higher thermotolerance and proofreading activity.
◦ Slower synthesis than Taq (has proofreading)
• Phusion polymerase
◦ Enzyme fused with processivity enhancing domain
◦ even higher delity
◦ High synthesis rate (because of processivity enhancing domain
TdT
• Eukaryotic enzyme that adds random bases to 3' end of single or double stranded DNA
• Does not require template to copy, just extends an existing 3' OH group.
• Can be used to join DNA by homopolymers tails
◦ Use TdT to add homopolymer tail onto target DNA
◦ Add homopolymers tail in to vector
◦ Allow recombination
RNA polymerases
• Unlike DNA polymerase which extend an existing DNA strand (ie. require primer or 3'OH), RNA polymerase start
de novo synthesis of RNA.
• Eubacterial type RNA (ie. E.coli RNA)
◦ multiple subunit couplex (2 alpha, 2 beta, and w subunit) with sigma factor.
◦ Learned previously in one of the lectures
• Bacteriophage type RNA (ie. T7, T3, and SP6 RNA)
◦ Single subunit enzyme with both promoter recognition and RNA synthesis activity.
◦ High speci city for promoter - very speci c for its promoter.
, ◦ High delity
Reverse Transcriptases
• it is a type of DNA polymerase (makes DNA) so it requires existing 3' OH group provided by an annealed primer.
• Makes cDNA from RNA strand or ss DNA.
• Requires primer that pair to the mRNA!
◦ Can be gene speci c primers
◦ oligodT priming - mRNA has poly A tail so add a bunch of T primers.
◦ Random hexameter priming
• Common reverse transcriptase used are from retroviruses (ie. AMV reverse transcriptase and MMLV reverse
transcriptase)
• Fidelity, thermostability and processivity
◦ Fidelity - viral enzymes are evolved to have high rate synthesis so there are higher error rates (no
proofreading activity)
◦ They are not very thermal stable.
◦ Processivity - how long enzyme stays until falling o from template
PCR
Requires
• Primers - recall DNA polymerase require primers
• Thermostable DNA polymerases
• Programmable thermocyclers
The position of the primers
• PCR involves synthesis of a product of a speci c size, de ned by the positions of the primers.
• For PCR, the primers are designed by the experimenter, synthesized chemically, and, by hybridizing to genomic
DNA, “tell” the polymerase which part of the genome to copy. As discussed in the previous section, DNA
primers (in essence, the same type of molecules as DNA probes but without a radioactive or uorescent label)
can be designed to uniquely locate any position on a genome.
• Primers mark the left and right boundaries of the DNA to be ampli ed. (You will have more than one type of primer in the
PCR
De ned Products
De ned product (the size wanted) is rst made in the third cycle. It increase exponentially
Products of unde ned size increases linearly.
fi
, The desired PCR product will be a duplex of the defined length fragment.
Wanted products are those that start and end at the primers.
Calculations
• Total number of strands = 2 x 2^n
• Molecules of de ned products = 2^n-2
◦ De ned products = double stranded product where both strands are of desired length
The maximum ampli cation rate/capacity is never reached
• only possible if substrates are unlimited and when enzyme is fully active
• Even at exponential phase, correct priming and complete strand synthesis is not 100%
• Later on, substrate starts to run out and enzyme slowly dies.
• So we do not achieve the full capacity of 2^n-2.
Primer Design
• Optimum length is 18-24 bases. Short enough for e cient binding, long enough for speci city.
• GC content 40-60^ (good for binding well to template). Last two bases are G or C. It is important to have last two
bases that bind well because the 3' OH end group is where the Taq polymerase depends on to synthesise
• Avoid sequences that allow primer primer annealing (check that the primers added into the solution do not
anneal together or it will make primer dimers)
• Tm of primers should be similar (<5ºC di erence) and between 60ºC and 75ºC.
◦ Having ™ similar means they are both e cient are same temperature
• Primer can be designed to have a primer tail that adds additional sequence to the end of the PCR product.
• Primer dimers are seen at the bottom of the gel (small size).
Amplicon
• Amplicon = the product
• Smaller amplicons are ampli ed more readily.
◦ This is also why primer dimers form readily because primers are short.
◦ Primer dimers are seen at the bottom of the gel (small size).
Annealing temperature
• Usually 5ºC below the ™ of the primer.
• This is e cient for anealing but minimizes o target binding
• O target binding tend to increase with lower temperatures.
Pcr applications
• Detect presence or absence of DNA
◦ Amplify by PCR and do gel electrophoresis or hybridisation etc
• Cloning: Amplify DNA
• Site directed mutagenesis by overlap PCR
◦ Primers designed with mutation
‣ 2 types of primer that will overlap and are complimentary in terms of the mutation (sense and antisense)
‣ The primers anneal to the same spot on the DNA but synthesize in opposite directions
◦ Primers anneal and PCR happens. PCR fragments produced
◦ Because the strands have a common site with complemetnary sequence, the strands can anneal
Introduce mutations in overlapping, complementary oligodeoxyribonucleotide (oligo) primers. These anneal to the same spot on the DNA and
will generate two DNA fragments with overlapping ends via PCR. These fragments are combined in a subsequent 'fusion' reaction in which
the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand.
The resulting fusion product is amplified further by PCR.
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