Techniques for DNA Amplification

Dr. Zeyad Akawi Jreisat M.D, M.A, Ph.D

Cloning genomic DNA in bacteriophage λ

Screening genomic libraries in bacteriophage λ

POLYMERASE CHAIN REACTION (PCR)

PCR

• The PCR technique is basically a primer extension reaction for amplifying specific nucleic acids in vitro.

• PCR will allow a stretch of DNA (usually fewer than 3000bp) to be amplified to about a million fold.

• The particular stretch of DNA to be amplified, called the target sequence

Composition of the reaction mixture

Template DNA:

• Usually the template DNA amount is in the range of 0.01-1ng for plasmid or phage DNA and 0.1-1µg for genomic DNA, for a total reaction mixture of 50µl.

• Higher template DNA amounts usually increase the yield of nonspecific PCR products.

PRIMERS

• A primer is a short segment of nucleotides that is complementary to a section of the DNA that is to be amplified in the PCR reaction. Primers are annealed to the denatured DNA template to provide an initiation site for the elongation of the new DNA molecule. Primers can either be specific to a particular DNA nucleotide sequence or they can be "universal." Universal primers are complementary to nucleotide sequences that are very common in a particular set of DNA molecules. Thus, they are able to bind to a wide variety of DNA templates.

Primers length should be from 15-30not less than 15 because it might be non specific, where multiple regions have the same sequence not more than 30 because it might anneal with each other

PRIMERS cont…

• Animal cell lines contain a particular sequence known as the "alu gene". There are approximately 900,000 copies of the alu gene distributed throughout the human genome, and multiple copies distributed through the genome of other animal cells, as well. Thus, the alu gene provides the sequence for a universal primer for animal cell lines. The alu primer is especially useful in that it binds in both forward and reverse directions.The alu universal primer seqeunce is as follows:5' GTG GAT CAC CTG AGG TCA GGA GTT TC 3' (26mer)

• When using universal primers the annealing temperature on the thermal cycler is lowered to 40-55 degrees C.

Other components

• dNTPs

• Taq DNA polymerase – heat resistant polymerase

• The Master mix buffer (Tris-HCL, pH 8.3, KCL,mM MgCl2)

Principle of the PCR

• The cycling reactions:

– Denaturation

– Annealing

– Extension

where you take the primers ? it depends,, if you know the DNA that you are going to amplify you will construct the primer based on the DNA sequence that you know" the primer's sequence that flank 5' and 3' region

if you don’t know the sequence we have to add the DNA of interest which is unknown to a plasmid that you know the sequence of the flanking region to use these flanking regions to form primers

Polymerase chain reaction

PCR

The exponential amplification of the gene in PCR

PCR Product at different T

Synthesis of cDNA from mRNA

Modification of cDNA for cloning

Directional cloning of foreign DNA

Production of transgenic animal