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DNA Techniques (PCR and

Sequencing)
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Date @26/10/2020 9:00 AM-10:00 AM

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Module BB MMB3001

💡 Check DNA/RNA concentration using a spectrophotometer at 260nm

(N.B. sample is NOT mechanically disrupted as DNA would break down
into small pieces and would be indistinguishable from RNA for the
spectrophotometer)

Principles of PCR
Uses DNA polymerase: Reads one sequence and makes a copy of it

Exponential amplification

Components: DNA polymerase, primers, buffer, MgCl2

DNA Techniques (PCR and Sequencing) 1

 Conditions (provided by thermal cycler): Denaturation (95 deg C), Annealing (50-
60 deg C), Extension (68-72 deg C); Cycles (25-40); Heated lid (103 deg C to
prevent the escape of reagent that evaporates)

Primer design
DNA is always written and read from 5' to 3' end

Forward and reverse primers (complement to the desired part of DNA), that
should have similar melting temperatures (difference <5 deg C)

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 PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length.
Two primers are used in each PCR reaction, and they are designed so that they flank the target
region (a region that should be copied). That is, they are given sequences that will make them
bind to opposite strands of the template DNA, just at the edges of the region to be copied. The
primers bind to the template by complementary base pairing. When the primers are bound to
the template, they can be extended by the polymerase, and the region that lies between them
will get copied.

Length of 13-25 bases

GC content is 40-60%, with an ideal of 50%

Melting temperature (Tm) between 55-65 deg C

GC clamp at the 3' end (since G-C bind more strongly, they are desired at the
end of the primer before DNA polymerase starts extension)

Pay attention to...

Avoid stretches of one nucleotide or repeats because polymerase is more likely
to make a mistake

Avoid regions which have secondary structure, as this leads to the formation of
hairpin loops or primer-dimers (test primers beforehand)

BLAST primers for gene specificity

Start PCR optimisation 3-5 deg C below the lowest melting temperature

Special rules
Add 5-6 "extra" bases if including a restriciton site (increases the efficiency of
enzyme binding)

To include a mutation in a primer, include it centrally

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 When designing sequencing primers, ass 50-100 bp to each end

Primers with 5' overhangs have much higher melting temperatures than just the
binding region

End-point PCR vs qPCR

Rules for designing qPCR primers

cDNA synthesis

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