DNA/RNA associated methods and their application
Polymerase chain reaction (PCR)
PCR is one of the oldest techniques (developed in 1983.) used for multiplying DNA sequences. DNA piece could be amplified thousand to million times. It’s used for identification of hereditary disorders, for paternity testing or DNA profiling (in forensics), for DNA -based phylogeny and for diagnostics of infectious diseases. DNA piece that needs to be multiplied is mixed with heat stable DNA polymerase (usually Taq polymerase), DNA primers and nucleotides in the buffer solution containing certain amount of Mg2+ (or Mn2+) and potassium ions. Initial step is DNA melting where high temperature is breaking double DNA strand in two pieces. After separation, DNA primers are attaching to the single stranded DNA and assembly could start. Each DNA piece serve as template for DNA polymerase that is adding complementary nucleotides from the solutions until new double helix is created. By rising the temperature, cycle can start all over again. It is usually repeated for 20-40 times and in each cycle amount of DNA product is doubled.
Quantitative PCR (qPCR) or real-time polymerase chain reaction
This method is simultaneously amplifying and quantifying produced DNA. Oligonucleotids in solution are labeled with fluorescent dyes that could be visualized after hybridizing with the complementary nucleotides on the DNA. This method is mostly used in research and for diagnostic purposes (for cancer, infectious diseases, flu…).
Reverse transcription polymerase chain reaction (RT-PCR)
In RT-PCR method, RNA molecule is reversely transcribed into its DNA complement (cDNA) using enzyme reverse transcriptase. cDNA is further amplified using PCR. This technique is common when viruses are under investigation, as most of their genomes are consisted of RNA.
Gel electrophoresis is used for separation of the molecules based on their size and electric charge. Porous gel is usually made of agarose or polyacrylamide. Potato starch could also be applied. When gel is placed in the electric field, movement of the negatively charged nucleic acids will depend on their size. Shorter pieces will move faster and pass greater distances on the gel compared to long pieces, allowing quick and efficient separation of the mixed population (RNA and DNA) of the nucleic acids. Visualization of the separated molecules is achieved using ethidium bromide for DNA and RNA (provides blue fluorescence under UV light), or silver stain or Coomassie Brilliant Blue dye for proteins. Main applications of gel electrophoresis are in PCR products analysis, in restriction mapping of cloned DNA, for the separation of the restricted DNA pieces prior some other laboratory analysis…
Macromolecule blotting and probing
method is used to detect specific DNA sequence in the sample. It combines gel electrophoresis (for DNA pieces separation) and probe hybridization (to determine if sample contains DNA complementary to the probe). Main applications are in homology based cloning, for identification of the methylated sites in certain genes or in knockout stem cell engineering.
is used to detect RNA in the sample. It provides information on gene expression patterns in different tissue/organs, in various developmental stages, during the course of the treatment….
, known as protein immunoblot, is used to detect protein in the sample. Specific antibodies are used for targeted proteins after they are separated on the gel. This method is applied for HIV, Bovine spongiform encephalopathy, Lyme disease or Hepatitis B infection testing.
DNA microarray (DNA chip, biochip)
This technique is used when multiple genes expression need to be measured simultaneously. Each gene spot contains specific DNA sequence (probes) that are used to hybridize cDNA or RNA (target) under specific conditions. Probe-target hybridization is detected by labeling the target with fluorescent dye. Array can contain thousands of genes and parallel experiments could be easily performed. This method can be used for genetic profiling, comparative genomic hybridization, single nucleotide polymorphism detection, selection of the appropriate drug candidate….
New technique in extracting genomic data resulted as collaboration between scientist from the Scripps Institution of Oceanography and The Skaggs School of Pharmacy. Raw biological material could be evaluated for the known and unknown peptides using novel mass spectrophotometer. After peptides are fragmented to their amino acid building blocks, scientists could map them to the genome level. This method is unique, as it allows tracking “biosynthetic pathways”, or how molecules are reassembled (all the way from genomic expression to the end-product development). Two new classes of peptides are discovered using this technique. Information on “biosynthetic pathways” is valuable in a lot of fields, especially in pharmaceutical industry.