Molecular Biology

PCR is an acronym which stands for polymerase chain reaction. What is a polymerase? A polymerase is a naturally occurring enzyme, a biological macro-molecule that catalyzes the formation and repair of DNA. The accurate repli-cation of all living matter depends on this activity. In the 1980s, Kary Mullisat Cetus Corporation conceived of a way to start and stop a polymerase’s ac-tion at specific points along a single strand of DNA. What is the chain reaction? Mullis also realized that by harnessing this component of molecular reproduction technology, the target DNA could be exponentially amplified. The PCR technique is basically a primer extension reaction for amplifyingspecific nucleic acids in vitro. The synthesis of DNA begins with binding ofprimers to the DNA template and single nucleotides (dATP, dCTP, dGTP &dTTP) are added to the 3’ end of the growing DNA molecule. The use of athermostable polymerase which tolerates the high temperature of up to 95oC in the denaturing step allows the dissociation of newly formed complimentary DNA and subsequent annealing or hybridization of primers to the target sequencewith minimal loss of enzymatic activity. Inputs for PCR includes: (1) Two oligonucleotides are synthesized, each complementary to the two ends of the DNA fragments to be amplified. They are used as primers. (2) Thermostable DNA polymerase TaqI. PCR consists of repeating a cycle with three phases 25-30 times. Each cycletakes about 5 minutes.Phase 1: The denaturing step of separating double stranded DNA by heat;Phase 2: Cool; add synthesis primers to anneal to the denatured DNA;Phase 3: Add DNA polymerase TaqI to catalyze 5’ to 3’ DNA synthesis.After the last cycle, Phase 3 is kept for a longer time at about 10 minutes toensure that DNA synthesis for all strands are complete. Then, only the flankedregion by the primers has been amplified exponentially, while the other regionsare not. PCR method is used to amplify DNA segments to the point where it can bereadily isolated for use. When scientists succeeded in making the polymerase chain reaction perform as desired in a reliable fashion, they had a powerful tech-nique for providing unlimited quantities of the precise genetic material for doingexperiment. Some examples of PCR applications are: (1) Clone DNA fragments from mummies; (2) Detection of viral infections.

Gel electrophoresis

Gel electrophoresis, developed by Frederick Sanger in 1977, is a method thatseparates macromolecules-either nucleic acids or proteins-on the basis of size,electric charge, and other physical properties.A gel is a colloid in a solid form. The term electrophoresis describes themigration of charged particle under the influence of an electric field. Electro refersto the energy of electricity. Phoresis, from the Greek verb phoros, means “to carryacross.” Thus, gel electrophoresis refers to the technique in which molecules areforced across a span of gel, driven by an electrical current. Activated electrodes ateither end of the gel provide the driving force. A molecule’s properties determinehow rapidly an electric field can move the molecule through a gelatinous medium. Organic molecules such as DNA are charged. DNA is negatively charged due to the high phosphate residues in their backbone. A gel is prepared which willact as a support for separation of the fragments of DNA. Holes are created in the gel. These will serve as a reservoir to hold the DNA solution. DNA solutions (mixtures of different sizes of DNA fragments) are loaded in a well in the gel. During electrophoresis, DNA migrates towards the positive electrode. The greater the charge on the DNA molecule, the faster it migrates. However, the movementis retarded by the gel matrix, which acts as a sieve for DNA molecules. Large molecules have difficulty getting through the holes in the matrix. Small molecules move easily through the holes. Because of this, large fragments will lag behindsmall fragments as DNAs migrate through the gel. As the process continues, the separation between the larger and smaller fragments increases. The mixture isseparated into bands, each containing DNA molecules of the same length. An application of gel electrophoresis is to reconstruct DNA sequence of length 500-800 within a few hours. The idea is to, firstly, generate all sequences end with A. Then we can use gel electrophoresis to separate the sequences end with A intodifferent bands. Such information tells us the positions of A’s in the sequence. Similarly, we can process for C, G, and T accordingly. During electrophoresis, the fragments move towards the positive end. The unknown DNA sequence is reconstructed from the relative distances of the fragments. The letters A, C, G and T at the negativeend refers to ddATP, ddCTP, ddGTP and ddTCP reation mixtures respectively. Usually, the obtained sequence is verified by carrying out the same sequencing method on the complementary strand. The generation of fragments ending in a particular base can be achievedthrough two methods, i.e. Maxam-Gilbert Sequencing or Sanger Method. InMaxam-Gilbert Sequencing, DNA samples are divided into four aliquots and fourdifferent chemical reactions are used to cleave the DNA at a particular base (A,C, G or T) or base type(pyrimidine or purine). In Sanger Method, DNA chainsof varying lengths are synthesized by enzymes in four different reactions. Eachreaction will produce DNA ending in a particular base.DNA sequencing can be automated by using laser to detect the separated products in real time during gel electrophoresis. The four bases are labelled with different fluorescence and they are placed in a single lane. In this way, many DNA samples can be sequenced at 1 time. This can sequence a total of 900 basesin length and achieve a 98% accuracy well beyond 900 bases.

Hybridization

Routinely, biologists need to find a DNA fragment containing a particular DNA subsequence among thousands of DNA fragments. The above problem can besolved through hybridization in the following steps:

1. Suppose we need to find a DNA fragments which contains ACCGAT.

2. Create probe which are inversely complementary to ACCGAT

3. Mix the probes with the DNA fragments

4. Due to the hybridization rule (A=T, C=G), DNA fragments which contain ACCGAT will hybridize with the probes

DNA Array

The idea of hybridization leads to the evolution of DNA array technology, whichenable the researchers to perform experiment on a set of genes or even the wholegenome. This completely changes the routine of one gene in one experiment andmake easier for researchers to obtain whole picture on the particular experiment. DNA array is an orderly arrangement of thousands of spots, each containingmany copies of the same DNA fragment. When the array is exposed to the target solution, DNA fragments in both array and target solution will match based onhybridisation rule: A matches to T while C matches to G through hydrogen bonds. Such idea allows us to do thousands of experiments at the same time.

Application of DNA Arrays

DNA arrays can be used for sequencing using its hybridization method. Thebasic idea is that if all the subsequences of the target sequence are presented inthe DNA array chip, after hybridization, the target sequence can be constructedfrom overlapping of oligonucleotides by algorithms from the spectrum of inten-sities generated at every spots. However, due to the presence of repetitive DNA that obstructs reconstruction, the method is unlikely to be used in sequencing complex genomes, but useful for short non-repetitive DNA fragments. The fab-rication of DNA chip can be done by combining the photolithographic methodand combinatorial synthesis of oligonucleotides on the surface of glass chips. DNA array can also help researchers to investigate the expression profile of a cell. Through spotting the complementary sequence of genes on the DNA chip,the activities within a cell can be monitored by observing the expression levels of these genes at different conditions or time points. Due to hybridization, we canalso measure the concentrations of different mRNAs within a cell.There are many other applications where DNA arrays can be put to good use.

More Advanced Application

Mass SpectrometryMass spectrometry (MS) is one type of instruments that is used for analyzing biomolecules, particularly proteins and peptides. MS is very important as a toolsin proteomics field. MS offer three types of analyses in Proteomics analysis. First, MS can provide highly accurate protein mass measurements compared tothe traditional gel electrophoresis (in this case, SDS-PAGE) which is not suffi-ciently sensitive. Secondly, MS can also provide accurate mass measurements ofpeptides from proteolytic digests. In contrast to whole protein mass measurements, peptide mass measurements can be done with higher sensitivity and mass accuracy. The data from this peptide mass can be searched directly against thedatabases, and frequently to obtain definitive identification of the target proteins. Finally, MS can also provide the sequence of peptides obtained from proteolytic digests. Indeed, MS is now considered the state-of-the-art in peptide-sequence analysis. MS sequence data provide the most powerful and unambiguous ap-proach to protein identification.