Amplifying DNA : The Polymerase Chain Reaction (PCR)

6.1.7. Amplifying DNA : The Polymerase Chain Reaction (PCR)

The conventional molecular cloning techniques may be considered in vitro DNA—amplifying tools. Interestingly, the latest development in the field of synthetic DNA* has evolved an altogether new method for the rapid amplification of DNA in vitro, broadly termed as the Polymerase Chain Reaction (PCR). In reality, PCR is capable of multiplying DNA molecules to the extent of a billion fold in vitro,

thereby giving rise to huge amounts of very specific genes employed for various purposes, such as : cloning, sequencing or mutagenesis. In short, PCR utilizes the enzyme DNA polymerase, which eventu- ally copies DNA molecules.

The polymerase chain reaction (PCR) for amplifying specific DNA sequences have been shown in Fig. 2.11. [Stage – A through Stage – F ]. These six stages have been duly explained here under :

A : The target genes (DNA — combinant form) if first heated to affect the separation of the strands of DNA ; secondly, a reasonably excess amount of two oligonucleotide primers**, of which one is complementary strand, is added along with DNA-polymerase ;

Stage –

B : As the resulting mixture attains the ambient temperature, the excess of primers relative to the target DNA makes sure that most target strands anneal to a primer exclusively and not to each other. In this way, the primer extension ultimately gives rise to a copy of the original double- stranded DNA.

Stage –

C : Further follow up of three above mentioned steps sequentially viz ; heating, primer annealing and primer extensions results into the formation of a copy of the original double-stranded DNA. In other words, DNA polymerase extends the perimers employing the target strands as a template.

Stage –

D : Another prime extension of the resulting product yields the second double-stranded DNA.

Stage –

* Synthetic DNA—short fragments of DNA of specified base sequence and widely used in molecular genetics. ** Primers : A molecule (usually a polynucleotide) to which DNA polymerase can attach the first nucleotide

during DNA replication.

PHARMACEUTICAL BIOTECHNOLOGY

Per-cycle

Target Genes [Nos : of Copies]

Target Genes

Polymerase Stage-A

Primer Extension

Stage-D

Repeat Cycle 3 8 Stage-E

Repeat Cycle 4 16 10 8

10 7 et

rg 6 10 a T

Stage-F f o ene 10 4 G 10 3

pies 10 2 Co

2 4 6 8 10 12 14 16 18 20 Nos of PCR Cycles

Fig. 2.11. PCR for Amplifying Specific DNA Sequences (Stage –A through Stage –F)

Stage – E : The end product obtained from the previous step is subjected to incubation for a suitable duration ; and the resulting mixture is heated once again so as to separate the strands. Subse- quently, the mixture is brought to the room temperature whereby the primers aptly get hybridized with the complementary regions of newly synthesized DNA. Thus, the whole process is repeated. In this particular instance, the two additional PCR-cycles give rise to 8 to 16 copies, respectively, of the origi- nal DNA sequence.

GENETIC RECOMBINATION

Stage – F : It represents a plot between the number of PCR cycles (along the X-axis) and the copies of the target gene (along the Y-axis). The graphical illustration depicts the effect of carrying out

20 PCR cycles on a DNA preparation initially having only 10 copies of a target gene. The resulting graph is semilogarithmic in nature.

Advantages of PCR-Technique

PCR-technique has two cardinal advantages, namely : (a) Each and every cycle virtually doubles the content of the original target DNA, and

(b) A 10 6 to 10 8 fold increase in the target sequence is actually achieved after a 20-30 PCR cycle run.

Human Clone :

Severino Antinori — a 53 year old Italian embryologist along with his fellow scientist Robert Edwards jointly produced the first Test Tube Baby and thereby created a history in the entire world. After their spectacular achievement in the field of modern advancement in embryology Antinori is ever ready to repeat the history by creating the world’s first human clone. Contrary to the extremely opposed ethical challenges, Antinori with the aid of first human in vitro fertilization (IVF) technique successfully enabled a 62 year old lady in 1994 to become the oldest to bear a baby. He feels very strongly and hence advocates vehemently that the very ‘technology of cloning’ is a logical and legitimate extension of IVF which may certainly help specifically the unfortunate infertile couple to have children.

After years of whole-hearted dogged and dedicated efforts the ‘scientists’ have ultimately suc- ceeded in developing the key techniques to ‘reset’ the DNA of living cells that critically possessed specialist functions ; and they behaved as if they were a newly fertilized embryo that eventually grew into a clone of the adult. In early 1998, the experts at the University of Hawaii carried out the successful

cloning of mice.

In the light of the above successful events the future prospect of ‘human cloning’ is now more or less perceived as a stark reality. Taking stock of the situation with regard to the astronomical develop- ment in biotechnology both the US government and the European Parliament have imposed and clamped most stringent and strictest legislations to outlaw its practice on the humans. In Great Britain, the strict control of the Human Fertilization and Embryology Authority (HFEA) that has not only made it abun- dantly clear but also strongly pronounced its clear cut objectives and intensions to completely block and reject outrightly any requests whatsoever to grant permission to carry out work related to human cloning.

Antinori argues and seeks support from the world body as well as the law enforcing countries to allow him to go ahead with human cloning —

‘‘What about the man who does not produce any sperm at all ? What should he do ? If he cannot reproduce himself why should he not reproduce his ’genes’ in this way — this is one of the few cases where it is acceptable to clone’’ .