AMPLIFICATION OF NUCLEIC ACIDS By Dr. Emad AbdElhameed Morad Lecturer of Medical Microbiology and Immunology
Amplification In vivo (inside a cell) In vitro (In a reaction tube) Genetic recombination Target amplification (PCR-based methods) Signal amplification Hot start PCR Nested Semi-nested Multiplex PCR RT-PCR Quantitative Branched chain DNA Conventional Real time
DEFINITION • In vitro method for amplification of a DNA segment (in a test tube). PCR was invented in 1983 by DR. Kary Mullis for which he received Nobel prize in chemistry in 1993.
PCR PCR tube THERMOCYCLER
REQUIREMENTS • DNA template: • The gene or DNA segment to be amplified. • Primers: • Small pieces of DNA formed of 15-30 base. • They are two primers (forward and reverse). • One primer complements the 3 ́ end region upstream of the gene to be amplified. • The other primer complements a region on the opposite strand downstream of the gene to be amplified. • The 3 ́ end of each primer points toward the target DNA.
DNA polymerase enzyme: • The polymerase enzyme used is called Taq DNA polymerase. • Taq means Thermus aquaticus bacterium. • It is heat stable enzyme (can function at the high temperatures). • This enzyme cannot copy DNA without primer. • dNTPs: • Deoxynucleotide triphosphates. • Four dNTPS are used: dATP, dTTP, dGTP, dCTP.
SO, PCR tube will contain: • DNA template • Forward and reverse primers • Taq DNA polymerase enzyme • dNTPs (dATP, dTTP, dGTP, dCTP) • Magnesium ion (co-factor for the polymerase enzyme) • PCR Buffer: to maintain pH and ionic strength of the reaction solution.
STEPS Denaturation Annealing Extension
Denaturation: • By heating the target DNA at 90-95 °C for 30-60 seconds, the double helix will be separated into two strands. • Annealing: • The temperature is then lowered to 50-65 °C. • At this temperature the primers anneal (hybridize) to their complementary sequences on the target DNA. • This takes about 20-30 seconds.
Extension (elongation): • At 70-75 °C, the polymerase enzyme adds nucleotides to the primer to make a copy of the template. • The 3 steps (denaturation & annealing & extension) constitute one cycle which takes less than 2 minutes. • At the end of the first cycle, the template is duplicated. • The cycle is then repeated 30 or more times. • After 30 cycles, 1 million copy of the target DNA is obtained.
Detection of the amplified DNA (amplicons) • Agarose gel electrophoresis: • The amplicons will be stained by ethidium bromide and visualized by ultraviolet light. • Southern blot: • After gel electrophoresis, the gel is blotted on nitrocellulose membrane. • Amplicons could be detected by specific labeled probes. • Enzyme linked immunosorbent assay (ELISA): • Amplicons are labeled with digoxigenin. • Digoxigenin labeled amplicons are detected using biotinylated specific probes immobilized on streptavidin coated wells. • Detection is done by observing color reaction after adding enzyme labeled anti-digoxigenin antibodies and substrate.
APPLICATIONS • Detection of pathogens: • Pathogens unable to grow in culture such as hepatitis B virus, hepatitis C virus. • Pathogens very slowly growing in culture such as Mycobacterium tuberculosis. • Pathogens dangerous to grow in culture such as Human immunodeficiency virus (HIV). • Early detection of cancer: • By amplification of DNA of sheded cells in urine (urinary bladder cancer) or stools (colon cancer).
Forensic medicine: • By amplification of DNA from a dried blood spot or hair follicle to trace the accused person for a particular crime. • Genetic diseases: • Prenatal diagnosis of genetic diseases by amplification of samples obtained by amniocentesis. • Anthropological application: • Ancient DNA from mummies preserved at museums can be amplified into million copies.