Computer Primer Design-Bioinformatics

July 30, 2019 Off By admin

Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is a very versatile gene amplification method that has brought a tremendous progress in molecular biology and genetics. It is an in vitro method of amplifying a desired DNA sequence of any origin hundreds of million times in hours. Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments. PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology.

Components of PCR
The PCR reaction requires the following components:

DNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample.

DNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70°C.

Oligonucleotide primers : Short pieces of single stranded DNA (often 20-30 base pairs) which are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.

Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis.

Buffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity.

PCR procedure
All the PCR components are mixed together and are taken through series of 3 major cyclic reactions conducted in an automated, self-contained thermocycler machine.

Denaturation :
This step involves heating the reaction mixture to 94°C for 15-30 seconds. During this, the double stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds.

Annealing :
The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds. This allows the primers to bind (anneal) to their complementary sequence in the template DNA.

Elongation :
Also known at extension, this step usually occurs at 72-80°C (most commonly 72°C). In this step, the polymerase enzyme sequentially adds bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′ direction. Under optimal conditions, DNA polymerase will add about 1,000 bp/minute.

The procedure involves cycle of steps in which the double-stranded target sequence is denatured, oligonucleotide primers bordering the region to be amplified are annealed and the primers are extended by thermo-stable polymerases and dNTPs.

In DNA amplification by PCR, a desired cDNA clone is synthesized using mRNA as a template. Suitable primers are used to hybridize to the corresponding sequences, and they are extended in a chain synthesis reaction by thermo-stable DNA polymerases, using the inserted sequence as the template. The PCR mixture contains DNA bases (four types) and two primers (~ 20 bases long). The mixture is heated to separate the target sequence and then cooled (annealing) to allow the (i) primers to bind to their complementary sequence on the separated strands, and (ii) the polymerase to extend the primers into the new complementary strands. Repeated heating and cooling cycles multiply the target DNA exponentially, since each new double strand separates to become two templates for further synthesis. The nucleotide that the polymerase attaches will be complementary to the base in the corresponding position on the template strand (e.g. if the adjacent template base is C, the polymerase attaches G). The polymerase chain reaction proceeds with two primers, bound to the opposite strands of the gene target, and their 3’-ends pointing at each other. The reaction is terminated by the incorporation of dideoxynucleotides. The resultant is a series of fragments of different lengths for each primer.

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The design of appropriate short or long primer pairs is only one goal of PCR product prediction. Other information provided by in silico PCR tools may include determining primer location, orientation, length of each amplicon, simulation of electrophoretic mobility, identification of open reading frames, and links to other web resources.

With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

Many software packages are available offering differing balances of feature set, ease of use, efficiency, and cost. Possibly the most widely used would be e-PCR,freely accessible from the National Center for Biotechnology Information (NCBI) website. On the other hand, FastPCR, a commercial application, allows simultaneous testing of a single primer or a set of primers designed for multiplex target sequences. It performs a fast, gapless alignment to test the complementarity of the primers to the target sequences. Probable PCR products can be found for linear and circular templates using standard or inverse PCR as well as for multiplex PCR. VPCR runs a dynamic simulation of multiplex PCR, allowing for an estimate of quantitative competition effects between multiple amplicons in one reaction. The UCSC Genome Browser offers isPCR, which provides graphical as well text-file output to view PCR products on more than 100 sequenced genomes.A primer may bind to many predicted sequences, but only sequences with no or few mismatches (1 or 2, depending on location and nucleotide) at the 3′ end of the primer can be used for polymerase extension. The last 10-12 bases at the 3′ end of a primer are sensitive to initiation of polymerase extension and general primer stability on the template binding site. The effect of a single mismatch at these last 10 bases at the 3′ end of the primer depends on its position and local structure, reducing the primer binding, selectivity, and PCR efficiency

Types of PCR
In addition to the amplification of a target DNA sequence by the typical PCR procedures already described, several specialised types of PCR have been developed for specific applications.

Real-time PCR
Quantitative real time PCR (Q-RT PCR)
Reverse Transcriptase PCR (RT-PCR)
Multiplex PCR
Nested PCR
Long-range PCR
Single-cell PCR
Fast-cycling PCR
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR
In situ PCR
Variable Number of Tandem Repeats (VNTR) PCR
Asymmetric PCR
Repetitive sequence-based PCR
Overlap extension PCR
Assemble PCR
Intersequence-specific PCR(ISSR)
Ligation-mediated PCR
Methylation –specifin PCR
Miniprimer PCR
Solid phase PCR
Touch down PCR, etc

Applications of PCR
Some common applications of PCR in various fields can be explained in following categories.
Medical Applications:
1. Genetic testing for presence of genetic disease mutations. Eg: hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism
2. Detection of disease causing genes in suspected parents who act as carriers.
3. Study of alteration to oncogenes may help in customization of therapy
4. Can also be used as part of a sensitive test for tissue typing, vital to organ transplantation
genotyping of embryo
5. Helps to monitor the gene in gene therapy

Infectious disease Applications:
1. Analyzing clinical specimens for the presence of infectious agents, including HIV, hepatitis, malaria, tuberulosis etc.
2. Detection of new virulent subtypes of organism that is responsible for epidemics.

Forensic Applications:
1. Can be used as a tool in genetic fingerprinting. This technology can identify any one person from millions of others in case of : crime scence, rule out suspects during police investigation, paternity testing even in case of avaibility of very small amount of specimens ( stains of blood, semen, hair etc)

Research and Molecular Genetics:
1. In genomic studies: PCR helps to compare the genomes of two organisms and identify the difference between them.
2. In phylogenetic analysis. Minute quantities of DNA from any source such a fossilized material, hair, bones, mummified tissues.
3. In study of gene expression analysis, PCR based mutagenesis
4. In Human genome project for aim to complete mapping and understanding of all genes of human beings.

Insilico PCR
In silico PCR refers to computational tools used to calculate theoretical polymerase chain reaction (PCR) results using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome.

In silico PCR refers to a virtual PCR executed by a computer program with an input of a pair or a batch of primers against an intended genome that is stored in a silicon media (e.g., a database server). In silico PCR aims to test PCR specificity including the target location and amplicon size in one or multiple target genome(s). It can identify the mismatches in primer binding sites due to known single nucleotide polymorphisms (SNPs) and/or unwanted amplicons from a homologous gene or a pseudogene. With the development of sequencing technology and rapid cost reduction, many genomes have been sequenced and annotated in databases. Such a wealth of genomic information makes in silico PCR possible. In silico PCR analysis can assist in the selection of newly designed primers and avoid potential problems before primer synthesis or a “wet bench” experiment. This analysis is also useful to validate the published primers before being blindly adopted.

Insilico PCR using NCBI Primer-BLAST
One critical primer property is the target specificity. Ideally, a primer pair should only amplify the intended target, but not any unintended targets. This is especially important for real time quantitative PCR (qPCR) where in many cases the amount of PCR product is represented by the total intensity of fluorescence incorporated into amplified DNA and any amplification of unintended targets can affect the measurement. Since different parts of chromosomes or transcripts may share some nucleotide similarity due to either homologous regions or fortuitous matches, it is not uncommon that a primer pair intended for one target will also bind to another one, resulting in non-specific target amplifications.In general, mismatches towards the 3’ end affect target amplification much more than mismatches towards the 5’ end. Given the variable effects of the mismatches and the likelihood that users may have different criteria based on their own experimental conditions, it is important that a software tool should offer the capability to detect up to a few mismatches over the entire primer range and the flexibility to change the specificity settings.

Primer-BLAST as a general purpose public tool that helps users design target-specific primers. Primer-BLAST offers flexibility to accommodate different primer design needs. Users can either design new primers or check the specificity of pre-existing primers. Notably, Primer-BLAST incorporates a global alignment mechanism and is designed to be very sensitive in detecting potential amplification targets. Finally, it has the capability to place primers based on exon/intron boundaries and SNP locations.

These tools are used to optimize the design of primers for target DNA or cDNA sequences. Primer optimization has two goals: efficiency and selectivity. Efficiency involves taking into account such factors as GC-content, efficiency of binding, complementarity, secondary structure, and annealing and melting point (Tm). Primer selectivity requires that the primer pairs not fortuitously bind to random sites other than the target of interest, nor should the primer pairs bind to conserved regions of a gene family. If the selectivity is poor, a set of primers will amplify multiple products besides the target of interes

Case study
we design primers using the human zinc finger protein 419 (ZNF419) transcript variant 5 mRNA (Genbank accession NM_001098494).
1.Enter GI: NM_001098494
2.The search used default values that require at least one primer (for a given primer pair) to have two or more mismatches to unintended targets in the last five bases at the 3’ end.
3.The specificity checking was performed against the NCBI RefSeq mRNA database with organism limited to human, since the goal was to find primer pairs that are specific to this transcript only among the human transcriptome.
4.To avoid possible genomic DNA amplification, the option “Primer must span an exon-exon junction” is selected.
5.Click get primers