Polymerase Chain Reaction ( PCR )

This is a method to produce very large numbers of copies of specific DNA sequences without cloning. Therefore PCR can amplify specific sequences or add sequences such as  endonuclease  recognition  sequences as primers to cloned DNA.

PCR consists of 5 main components.

-Target DNA

-Single stranded Oligonucleotides (primers)

-d NTPs (dATP, dCTP, dTTP, dGTP)

-Taq DNA polymerase

-Termocycler

There are three main steps in PCR.

Step1:  Denaturation. The mixture of excess primer and DNA fragment is heated to about 95° C causing the double strands of target DNA to be denatured into single strands.

Step 2: Annealing of Primers. The temperature is dropped down between about 35⁰- 65°C

As the temperature decreases the single strands of DNA reassociate into double strands. Large excess of primer allows two primers anneal or bind to their complementary sequences on the target DNA leaving the rest of the fragment single-stranded.

Step 3: Primer Extension. The temperature is raised to 70⁰- 75°C Taq  polymerase is added. Taq polymerase extends the primer into a complementary copy of the entire single-stranded fragment, in the 5’-3’ direction.  As both the DNA strands are replicated, two copies of the original fragment are gained.

This process is repeated many times. At each time, the number of DNA copies doubles. This is continued until enough copies are gained for the analysis. 

Differences between physisorption and chemisorption

Different kinds of bacteriophages (phages)

M13

Filamentous.

About 870nm in length and 6nm in width.

Consists of single stranded DND (ssDNA).

Three kinds of capsomeres build the capside.

Infects E. coli by adsorbing to the cell and entering through F pilli. Therefore only infect F⁺ and HFr cells. Also male specific.

Does not kill the host. Particles are released by budding, therefore when the particles are released, the host cell is alive.

An efficient vector in gene cloning as it can hold longer pieces of foreign DNA.

T phage

Structure is composed of icosahedral head, double stranded DNA and a tail.

Infects E.coli

 Main types of T phages are T2, T4 and T12.

In the infection linear DNA of the phage is released to the host cell and becomes circular by replicating that later produces a long DNA chain known as ‘Concatamen’. This coils into the phage’s head by headfull mechanism while packaging.

Infection kills the host cell as the new particles are released outside by bursting the host cell.

Lambda (λ)

Composed of head and tail.

Head is consisting of double stranded linear DNA.

At both 5’ ends of the DNA strand, 12 complementary base pair, single stranded segments are present. These two ends are known as “cos ends”.

Because of the cos ends, phage chromosome circularizes before replication. Concatamen is produced during the replication and during the packaging, Terminase enzyme cuts off the cos ends.

Host cell is E.coli.

MS2

Contains the smallest known genome.

Super coiled single stranded DNA.

Infect only through sex pilli. Therefore male specific.

Infect E.coli.

Phi×174(ΦX174)

Contains a single stranded circular DNA.

After adsorption, synthesizes the complementary strand and becomes double stranded.

Use as a positive control in DNA sequencing.

G4

Structurally similar to ΦX174 phage.

Can infect susceptible E.coli cells.