Thoughts on economics and liberty

Notes on the science of PCR tests

This post supplements my other posts – it is a kind of “deep dive” (not too deep) into the science behind the tests, for my personal understanding of the issues involved.

MY OTHER POSTS ON PCR TESTS

Studies regarding the unreliability of PCR tests

A Portuguese court’s ruling against improper use of PCR tests

Complaint by Danielle Burnie to the TGA of Australia re PCR tests

NOTES TO BETTER UNDERSTAND THIS TECHNOLOGY

Genomics by John Archibald

The field of ancient DNA research began taking shape in the 1980s, concomitant with—and indeed made possible by—the development of a laboratory technique called the Polymerase Chain Reaction (PCR). Invented by the American Nobel laureate Kary Mullis, PCR uses a polymerase enzyme, a pair of short DNA ‘primers’ (needed by the polymerase to initiate DNA synthesis), and dNTPs to exponentially amplify a DNA fragment of interest from a complex DNA sample. PCR is used in a wide range of applications, everything from paternity testing and crime scene investigations to the study of microbial ecology (see Chapter 6). For ancient DNA research, what makes PCR so powerful is that it allows one to study the tiny amounts of DNA that linger in biological remains such as desiccated tissue, teeth, and bone. Once amplified using PCR to manageable quantities, ancient DNA can be sequenced and compared to that of present-day organisms using standard bioinformatic techniques. Ancient DNA sequences are the molecular equivalent of a time machine; they can teach us about the biology of organisms that no longer exist.

Extraordinary measures must be taken to ensure that ancient DNA sequences are truly derived from the organism of interest, rather than from the person who extracted the DNA, from the palaeontologists who handled the fossil, or from the zoo of microbes on and within the fossil at the time it was collected (these microbes can themselves be ancient or modern). The sensitivity of PCR is such that even trace amounts of modern DNA contaminating lab equipment and reagents can yield PCR products that can be mistaken for ancient ones. Even airborne DNA can be amplified and sequenced if it finds its way into PCR reaction vessels.

a limitation of early next-generation technologies was short sequence read lengths, initially on the order of fifty nucleotides. Depending on the size and complexity of the target genome, this can pose a serious challenge for accurate genome assembly.

Genetics from Genes to Genomes – Hartwell et al

PCR amplifies specific regions of DNA defined by two
oligonucleotide primers. Repeated cycles of synthesis
increase exponentially the number of copies of the target
DNA region.

The primers must be complementary to opposite
strands and have 5′-to-3′ polarities that point toward each
other through the region of interest. In practice, PCR is inefficient
if the primers are far apart, so the protocol generally
cannot amplify DNA regions greater than 25 kb long.

Essential Genetics and Genomics facebook.com/LinguaLIB

polymerase chain reaction (PCR) Repeated cycles of
DNA denaturation, renaturation with primer oligonucleotide
sequences, and replication, resulting in exponential
growth in the number of copies of the DNA sequence
located between the primers

It is also possible to obtain large quantities of a particular
DNA sequence merely by selective replication. The
method for selective replication is called the polymerase
chain reaction (PCR), and it uses DNA polymerase
and a pair of short, synthetic oligonucleotides, usually
about 20 to 30 nucleotides in length, that are complementary
in sequence to the ends of the DNA sequence
to be amplified and so can serve as primers for strand
elongation. Starting with a mixture containing as little
as one molecule of the fragment of interest, repeated
rounds of DNA replication increase the number of molecules
exponentially. For example, starting with a single
molecule, 25 rounds of DNA replication will result in
2^25 = 3.48 x 10^7 molecules. This number of molecules
of the amplified fragment is so much greater than that
of the other unamplified molecules in the original mixture
that the amplified DNA can often be used without
further purification. For example, a single fragment of
3000 base pairs in E. coli accounts for only 0.06 percent
of the total DNA in this organism. However, if this single
fragment were replicated through 25 rounds of replication,
99.995 percent of the resulting mixture would
consist of the amplified sequence.

The oligonucleotides act as primers
for DNA replication because they anneal to the ends
of the sequence to be amplified and become the substrates
for chain elongation by DNA polymerase. In the
first cycle of PCR amplification, the DNA is denatured
to separate the strands. The denaturation temperature
is usually around 95°C. Then the temperature is
decreased to allow annealing
in the presence of a vast
excess of the primer oligonucleotides. The annealing
temperature is typically in the range of 50°C to 60°C,
depending largely on the G 1 C content of the oligonucleotide
primers. The temperature is raised slightly,
to about 70°C, for the elongation of each primer. The
first cycle in PCR produces two copies of each molecule
containing sequences complementary to the primers.
The second cycle of PCR is similar to the first. The
DNA is denatured and then renatured in the presence
of an excess of primer oligonucleotides, whereupon the
primers are elongated by DNA polymerase; after this
cycle there are four copies of each molecule present in the original mixture. The steps of denaturation,
renaturation, and replication are repeated from 20 to
30 times, and in each cycle, the number of molecules
of the amplified sequence is doubled. The theoretical
result of 25 rounds of amplification is 225 copies of each
template molecule present in the original mixture.

polymerase because it was originally isolated from the
thermophilic bacterium Thermus aquaticus.
PCR amplification is very useful for generating large
quantities of a specific DNA sequence. The principal limitation
of the technique is that the DNA sequences at the
ends of the region to be amplified must be known so that
primer oligonucleotides can be synthesized. In addition,
sequences longer than about 5000 base pairs cannot be
replicated efficiently by conventional PCR procedures.
On the other hand, there are many applications in
which PCR amplification is useful. PCR can be employed
to study many different mutant alleles of a gene whose
wildtype sequence is known in order to identify the
molecular basis of the mutations. Similarly, variation in
DNA sequence among alleles present in natural populations
can easily be determined using PCR. The PCR
procedure has also come into widespread use in clinical
laboratories for diagnosis. To take just one very important
example, the presence of the human immunodeficiency
virus (HIV), which causes acquired immune deficiency
syndrome (AIDS), can be detected in trace quantities in
blood banks by means of PCR using primers complementary
to sequences in the viral genetic material. These and
other applications of PCR are facilitated by the fact that
the procedure lends itself to automation—for example,
the use of mechanical robots to set up the reactions.

 

 

Target DNA is limited to 20 kilo bases (human DNA has 3000 Mega bases)

(see from 50 seconds)

Illustrative: 70% or more positives can easily be false

In Hindi: a range of limitations outlined from 9:15

In Hindi: a discussion of limitations from 2:50

In Hindi from 8:40 about limitations

A discussion of what could go wrong with testing

 

Sanjeev Sabhlok

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