Download Ravi Sundaram What is PCR Why is it such a major breakthrough?

PCR
Ravi Sundaram
What is PCR
PCR stands for polymerase chain reaction. PCR is a technique to generate millions of copies
of a particular DNA sequence from a single piece of DNA. The words “chain reaction”
accurately capture the essence of the technique. In a nuclear chain reaction one nucleus splits
emitting particles that then cause two other nuclei to split which in turn cause four and then eight
and then sixteen… setting off an exponential explosion, literally. Similarly PCR creates an
explosion of copies. “Polymerase” is an enzyme that enables the chain reaction and we will
explain its role in greater detail in what follows. At its heart PCR is actually an algorithm. And it
is possible to understand how PCR works without understanding all the deep biology and
chemistry underlying it.
Why is it such a major breakthrough?
PCR is such a major breakthrough that biochemists and molecular biologists refer to biology as
essentially consisting of two epochs – before PCR and after PCR. Before PCR was invented
creating copies of DNA sequences involved painstaking and back-breaking lab work. Numerous
hours had to be spent with reagents in test-tubes to splice longer sequences and then separate the
desired portion using centrifuges and mass spectrometers. But all this work resulted in a very low
yield
PCR was “invented” (demonstrated) by Kary Mullis on 16 December 1983 while working as a
chemist at (the now defunct) Cetus Corp. PCR is now so common-place that it is considered as
indispensable and ordinary as the test-tube. It is utilized for a wide variety of application. These
include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the
diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic
sciences and paternity testing); and the detection and diagnosis of infectious diseases. When
detectives on CSI nail a suspect by lifting his DNA prints from a drinking glass - that’s PCR in
action.
In 1993, Mullis was awarded the Nobel Prize in Chemistry (along with Michael Smith) for his
work on PCR.
Why is it controversial?
Almost as soon as news of the discovery of PCR hit the scientific establishment, controversy
erupted. According to Mullis it was in the spring of 1983 while driving on the highway late one
night with his girlfriend that he got the idea for PCR. Cetus awarded him $10,000 for the patent
filing. A spate of lawsuits followed with Du Pont ultimately losing out to Roche which acquired
Cetus for three hundred million dollars. Academics pointed out that a 1968 paper of Hargobind
Khorana (another Nobel prize winner) and Kjell Kleppe had foreseen many of the central points
of the discovery. Senate committees were convened to debate whether PCR was really a
discovery at all and whether a patent was justified for something in which no new biology or
chemistry was invented.
The main reason for all the controversy was the enormous amount of money at stake. Another
factor in the controversy was the personality of Kary Mullis, which is/was quirky to say the least.
After his PhD from Berkeley he quit science to pursue a career as a sci-fi writer before returning
to Cetus as a biochemist. He has openly promoted fields such as astrology, in addition to being
an AIDS denialist and a climate change denialist. He has claimed to talk to extra-terrestrials. He
has decried establishment science and lab work claiming that he did his best work while surfing
or driving.
Basic biology
We need to know just a minimal amount of biology to follow PCR. In particular we need to
know about DNA. Deoxyribonucleic acid (DNA) is a nucleic acid containing the genetic
instructions used in the development and functioning of most known living organisms. The DNA
segments carrying this genetic information are called genes. DNA consists of two long polymers
of simple units called nucleotides. These two strands run in opposite directions to each other.
Each nucleotide contains one of four types of molecules called bases (or nucleobases or nucleic
acids). These four bases are denoted: A (adenine), T (thymine), C (cytosine) and G (guanine). (It
is the sequence of these four bases along the backbone that encodes information. This
information is read using the genetic code, which specifies the sequence of the amino acids
within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA
in a process called transcription.)
For our purposes we can think of a strand of DNA as consisting of a directed sequence of bases:
A→T→T→C→A→G→T
Within cells DNA is organized into long structures called chromosomes characterized by the
now-legendary Watson-Crick double-helix structure comprised of two anti-parallel strands with
the weak bonds: A –T and C–G. The above single strand is part of the following double-stranded
DNA:
A→T→T→C→A→G→T
| | | | | |
|
T←A←A←G←T←C←A
Elementary transformations
Rather than present Mullis’ discovery right away we are going to present the basic operations on
DNA that were well-known at the time of the discovery.
First, when a test tube of double stranded DNA is heated (in a controlled fashion) then it breaks
up into two constituent and complementary single strands:
A→T→T→C→A→G→T
| | | | | |
|
T←A←A←G←T←C←A
breaks into
A→T→T→C→A→G→T
and
T←A←A←G←T←C←A
Second when single strands of DNA are cooled then complementary copies (if sufficient in
proportion) will rejoin and form the complete double stranded DNA. This is the reverse of
heating.
Third, when a single strand of DNA is cooled in a test tube with an abundant supply of bases
along with polymerase then polymerase will create a complementary strand by moving along
the given strand and adding bases one by one. Thus for example, polymerase could start adding
to the following strand as follows:
A→T→T→C→A→G→T
becomes
A→T→T→C→A→G→T
| |
C←A
Note that polymerase will only add in the complementary direction to the given strand, in other
words, if polymerase started adding in the middle then it could not complete the initial part:
A→T→T→C→A→G→T
| | |
T←A←A
Further, note that complementary strands started by polymerase are not stable until they are
many (tens of) bases long. This means that the addition of polymerase will only rarely result in a
complementary strand that starts at the beginning and goes all the way to the end. More typically
it would start somewhere in the middle and continue to the end.
Lastly, it is possible to get quantities of short DNA strands (tens of bases) called primers
through conventional (pre-PCR) techniques.
The PCR algorithm
So here is the problem: given a small quantity of DNA containing a DNA (sub)sequence of
interest, create a large number of copies of the DNA sequence.
And at this point, knowing about DNA, heating, cooling, polymerase and primers you know
everything that Kary Mullis knew at the time of his revolutionary discovery.
Here is the solution to the problem. Let the given double strand be
……..→ο→……..→ω→…….
|
|
……..←ό←……..←ώ←…….
and let the sequence of interest be bracketed between ο and ω, i.e. ο→……..→ω.
Mullis’ brilliant idea was to add enough primers ο and ώ and then to heat and cool repeatedly in
cycles. Observer what happens after the first heating:
……..→ο→……..→ω→…….
and
……..←ό←……..←ώ←…….
Now comes the magic of polymerase with the primers, and we get:
……..→ο→……..→ω→…….
|
|
……..←ό←……..←ώ
and
ο→……..→ω→…….
|
|
……..←ό←……..←ώ←…….
After the second heating:
……..→ο→……..→ω→…….
and
……..←ό←……..←ώ
and
ο→……..→ω→…….
and
……..←ό←……..←ώ←…….
So far we are getting more than our desired sequence (or its complement) we are getting
extensions of our desired sequence (or its complement). Let us denote the desired sequence or its
complement by D. Let us denote a sequence which extends the desired sequence (or its
complement) on both ends by _D_ and let us denote a sequence which extends the desired (or its
complement) on only one end by D_. In other words we use _D_ to denote both:
……..→ο→……..→ω→…….
and
……..←ό←……..←ώ←…….
We use D_ to denote (both):
……..←ό←……..←ώ
and
ο→……..→ω→…….
We use D to denote:
ο→……..→ω
and
ό←……..←ώ
Observe that after the first heating we have 2 _D_s. After two heatings we have 2 _D_s and 2
D_s.
Once again the magic of polymerase with the primers, and it is after the third heating that we get
two copies (or complements) of the desired sequence. After the third heating we get 2 _D_s, 4
D_s and 2 Ds.
If we create a table of tallies of the three kinds of sequences we get:
Heating cycle
1
2
_D_
2
2
D_
0
2
D
0
0
3
4
5
2
2
2
4
6
8
2
8
22
n
2
2(n-1)
2^n-2n
Observe that each row totals to 2^n as it should. Observe also that the number of _D_s is
constant while the number of D_s grows linearly (or polynomially) while the number of the
desired sequence D grows exponentially.
Below we plot a tally of the three kinds of sequences to get a sense for why exponential growth
is so explosive.
Exponential vs Polynomial vs Constant
120
100
80
_D_
60
D_
D
40
20
0
1
2
3
4
5
6
7
References
1. Wikipedia, http://www.wikipedia.com
2. Lipton’s blog, http://rjlipton.wordpress.com/2009/08/13/amplifying-on-the-pcr-amplifier/