Polymerase Chain Reaction

Recombinant DNA Technology

Restriction Enzymes/ Restriction Endonucleases They restrict invasion by cutting at sites within the foreign DNA.

It recognizes specific sequences and cut both DNA strands. This specific base sequence is known as the "recognition sequence". Recognition sequences are palindromes.Example:EcoRI

5 GA A T T C 3

3 C T T A AG 5

Once the cuts have been made, the resulting fragments are held together only by the relatively weak hydrogen bonds that hold the complementary bases to each other. The weakness of these bonds allows the DNA fragments to separate from one each other. These are called sticky ends.

Ligase enzyme is then used to join the phosphate backbones of the two molecules.

Recombinant DNA - DNA molecule containing DNA fragments (from different sources) that are not naturally found

Vectors

Fragments produced after restriction enzymes cannot easily enter host cell

Gene carrier; also known as cloning vehicle

Characteristics of a Vector

It must be small, and unlikely to degrade during purification

It has markers (such as antibiotic resistance) that can indicate (in culture) whether transformation has been successful

It must have an origin of replication so that DNA can be replicated by the host cell's machinery

It has have several unique restriction sites so that the vector DNA will be cut only in the desired location, and that several such locations will be available for insertion of foreign DNA

Steps in Recombinant DNA Technology

1. Isolation of DNA

2. Cleaving of DNA with restriction enzymesA restriction endonuclease is used to cleave the source DNA into fragments3. Formation of recombinant DNAThe fragments of DNA are inserted into plasmids or viral vectors which have been cleaved with the same restriction endonuclease as the source DNA4. Introduce recombinant DNA to host cellsThe plasmids or viruses serve as vectors that can introduce the DNA fragments into cells

5. Propagation of clones

As each cell reproduces, it forms a clone of cells that all contain the fragment-bearing vector.Polymerase Chain Reaction

1. It is first developed by Kary Mullis in 1983

2. A more convenient method of amplifying specific DNA fragment.

3. Allows investigation of minute samples of DNA

4. It allows you to carry out in vitro multiple replications of target DNA

5. Can produce millions of copies DNA fragments from a single template

6. PCR is very specific

Components of PCR tubes

1. DNA template, which contains the region of the DNA fragment to be amplified

2. Two primers, which determine the beginning and end of the region to be amplified (see following section on primers)

3. DNA polymerase, which copies the region to be amplified

4. Nucleotides (dNTPs), from which the DNA polymerase builds the new DNA

5. Buffer, which provides a suitable chemical environment for the DNA polymerase

Instrument for PCR

The PCR reaction is carried out in a thermocycler, an instrument that automatically controls and alternates the temperatures for programmed periods of time for the appropriate number of PCR cycles (usually bet 30 - 40 cycles)

Three Steps in PCR1. Denaturation at >90C

The DNA template is heated at 95oC, breaking apart the two strands of the DNA double helix.

2. Annealing at 54C

The temperature is cooled at around 54oC, and the oligonucleotide primers designate the boundaries of the DNA strand being duplicated.

3. Extension at 72C The Taq DNA polymerase functions to add the corresponding nucleotide bases to the DNA strand. The result is two identical copies of the DNA strand.

These three steps correspond to one PCR cycle. This will be repeated, each time doubling the number of copies of the DNA strand in the mixture. After 30 PCR cycles, over one million copies of DNA strand can be made.Applications of PCR

1. For rapid diagnosis of infectious diseases

2. Detection of rare pathological events such as mutation leading to cancer

3. Examine DNA of extinct species (mammoth, dinosaurs)

4. In criminal investigations, DNA fingerprints are prepared from the cells in a tiny speck of dried blood or semen or at the base of a single human hair

5. Physicians can detect genetic defects in very early embryos by collecting a sloughed off cells and amplifying DNA