Challenge in PCR

PCR has become the most used technique in several field of molecular biology and diagnostic within few years since its invention. The majority of DNA fragment can be amplified without any problem; nevertheless, some regions into the genome are not so easily replicated, such as many regulatory domains, promoters, enhancers or other control elements. The main reason for all these difficulties is the high GC content that causes the formation of intramolecular stem loops during the initial cycles resulting in lack of amplification.

The slowdown PCR is a novel technique characterized by the use of slow ramping rates and an increased number of cycles at the same annealing temperature. Furthermore the addition of dc7GTP to normal dGTP in a ratio 1:3 maximally improves the reaction. Crucial step to do slow down PCR is to find an instrument –old- that uses slow ramp; in contrast modern instruments usually work at 5°C/sec to cool temperature. Then, also the choice of polymerase can contribute to obtain good results, this method may be limited only by the half- life of polymerase. However, Taq polymerase usually gives a positive amplification. In conclusion, the slow down PCR allows continuing important studies about gene expression regulation, by cloning DNA control sequence.

Application of molecular biology to medicine

Modern medicine is based on evidence. Despite few years ago, physicians based their diagnosis and therapies on their previous experiences, now clinical trials, approved protocols and worldwide accepted treatment help physicians to make the better choice for their patients. To reach this important goal, molecular medicine gave a big contribution. Molecular medicine is a novel branch of medicine in which molecular biology or biochemistry techniques are usually used to accomplish exams and screening or diagnose the diseases. Molecular biology labs have worked as research and development department to improve protocols or set up novel methodologies at the beginning just to be used for research purposes, then applied to diagnosis. For instance, we could focus our attention on the polymerase chain reaction, namely the PCR.
This techniques has been developed to produce DNA portion in vitro and has quickly revolutionized molecular biology, allowing cloning, sequencing and in general gene manipulation. The improved type of PCR, the real time PCR is now currently used in modern hospital to detect certain diseases, such as recidivate leukemia in patients’ blood or gene signature in familial diseases. Other important tools which have found a good application in diagnosis are the monoclonal/ polyclonal antibodies. Several techniques employed antibodies to detect proteins in cellular lysates through western blotting, or directly in whole cells, through the immuno-chemistry and molecular imaging. As well as, it’s also possible to purify small amount of native protein through immuno-precipitation and so on. Antibodies are really largely used in biochemistry labs and some techniques are applied also in diagnostic labs.
For instance, we can talk about the ELISA assay, normally used to quantify serum proteins or cell sorting analysis, in which some antibodies that specifically recognize surface proteins are used to separate different kind of cells. Finally, also cellular biology gave important results in modern medicine. The capability to culture in vitro cellular populations changes the therapeutic opportunity for a lot of diseases. For instance, now it’s possible to select healthy cellular population in leukemic patient, propagate it in vitro and then draft it, reducing in this way several problems of rejection and immuno-suppressive events. We can continue to talk about other examples of molecular, biochemical or cellular protocols that have found a great application in clinics. This overview strongly confirms how scientific advances are important because they have an immediate benefit on modern medicine and as a consequence on the human life. It’s important to remember that not only good protocols, but also good quality of science and good data management and reporting are two further parameters to improve modern medicine, but this is the topic for another post!!!

New method to quantify rare single-nucleotide polymorphism

Recent studies correlate rare allelic variants to many complex traits and combined effects of this deleterious mutations could explain susceptibility to many common diseases. To identify rare variants it’s necessary to genotype high number of individuals, by sequencing, or a pooled sample to minimize costs.

Druley and co-workers proposed a combination of molecular biology and computational analysis to achieve targeted resequencing and rare-variant detection. Procedure required PCR-amplification and sequencing with Illumina. They found that the first 12 bases of each Illumina read contained significantly fewer errors than later and, so, they used only this portion for their analysis.
They developed a new algorithm based on large deviation theory, named SNPSeeker. This program uses a second-order dependency error model for single-nucleotide polymorphism identification and considers the position of sequencing read (PCR cycle number) and the identity of two upstream bases. Thus, they improved the specificity of SNP calling and obtained results comparable to Sanger sequencing data, consistently reducing costs.
An important application of this method, is the combination of pooled-sample sequencing with genomic selection strategy to perform a more systematic survey of protein-coding DNA. This knowledge would be an important achievement for disease screening and tailoring risk-appropriate therapy.