The PCR is a process employed to amplify the DNA and used in DNA sequencing as well to get DNA copies, reduce contamination, and identify DNA mutations, and recombinant clones. All this happens in a small tube called PCR tube (what are PCR tubes).
PCR also known as the polymerase chain reaction (PCR) is used for nucleic acid amplification. It is a technique wherein the amplified product is denatured by using primers, followed by hybridization with a specific probe, generating an amplified product that can be sequenced. Nucleic acids are biological molecules that contain genetic sequences, which vary in size from about 18-24 kb. This technique involves a series of steps:
The target DNA molecule or RNA molecule (typically between 0.1–1 kb) is first prepared (usually labeled as a primer or probe)
A primer or Probe (also called a probe) usually contains a sequence of primers or probes, which help in amplifying the target molecule, followed by hybridizing these products with other complementary oligonucleotides, resulting in a final result (primer or probe)
To make this process easier and cheaper, the most important part is to select the appropriate number of cycles and temperature conditions required. There is a wide range of kits available; the commonest ones include Power SYBR Green Super High Capacity, EvaGreen, TaqMan Gold, Platinum II qPCR Kit, ABI Prism Real-Time PCR, Maxisorberge, and iStar PCR systems. These kits can be purchased from different companies such as Agilent, Invitrogen, QIABULGENE, Danaher, Quidel, etc.
The target genes can be isolated (isolated from the sample) from some organisms like bacteria, yeast, viruses, or plants. The target gene fragments can then be ligated to avoid the loss of their sequences
In order to have a single band of approximately 50 bp in length (short fragment), the PCR should be carried out using primers/probes, with the use of a relatively low cycle (low cycle number). However, the method is not suitable to detect the presence of multiple bands of smaller sizes (short fragments). Thus, it is important to perform the whole process of PCR at least twice, after each cycle (if there are more than two PCR runs, using lower numbers of cycles would increase the cycle number).
DNA samples (such as plasmids, rRNA, genomic DNA, etc.) can be amplified using single-stranded PCR, single-stranded RT-PCR, and double-stranded RT-PCR, respectively. Amplification of single-stranded cDNAs using single-stranded PCR is also possible, however, only when using a probe that has a single strand. On the other hand, double-stranded RT-PCR using one specific primer/probe can be performed at any number of cycles depending on the probe type.
Types of PCR and its Application in Sequencing
PCR (polymerase chain reaction) is a laboratory-based technique using sequences from one species, as templates. After the sequences are ligated to the same probes with no loss, they are then used to amplify other sequences that are similar to them.
PCR can be used in numerous applications from genotyping to genetic analysis to evolutionary studies, including evolution analysis. It enables the detection of specific alleles and variants present in the genome sequences, thereby enhancing understanding of the genetics of the host organism.
PCR can also be done using primers/probes (called ‘in situ primers) or primers/probes (called ‘in situ primers). Primers and probes can be used in various manners but are mostly used as follows:
Primers can be used using RT-PCR, which employs both reverse transcription (RT) and PCR. In RT-PCR, the reverse primer can serve as primer along with the forward primer. For instance, primers can be used to detect the insertion/deletion events that occur in bacterial genomes or the insertion/deletion events that occur in human genes. Using RT-PCR, several primers can be used to determine whether a particular fragment is present in the amplified product, or whether an insertion/deletion event is present. If a fragment is present, the target fragment is then sequenced using the corresponding primer.
Primers may also be used for qualitative RT-PCR to investigate whether fragments present in the amplified product are identical to those present in the reference fragment. For example, using the 16S rRNA, it may be possible to detect rRNA deletion and insertion/deletion events. As another possible application, a combination of non-coding primers and probes may be used to test the quality of the input sequence (e.g., to check for the presence of insertions/deletions or to test the efficiency of restriction digestion), fragments present in the amplified product can be sequenced using the appropriate probe (primer/probe).
In some cases, PCR can also be used for the detection of point mutations. Multiple fragments can be detected via single-stranded PCR and amplified products can be sequenced using the corresponding probe (primer/prob). Notably, even if single fragments are sequenced, it would not be possible to distinguish them at a low level of specificity because of the short length. Therefore, multiple fragments should be sequenced after each cycle using a probe with a longer length.
In some instances, PCR can also be used to prepare templates from small pieces of DNA. Single-stranded PCR can also be used to isolate and sequence fragments present in the template
Another interesting feature of PCR is its ability to facilitate the identification of certain diseases, as revealed through multiple tests carried out over the same set of samples, so as to accurately diagnose disease. Such multiple tests are called PCR-Sequencing tests. They are typically applied to the detection and differentiation of pathogenic bacteria, toxins, and viruses in biological samples, while others are meant as diagnostic tests. This capability can be used to identify pathogens such as HIV (Human Immunodeficiency Virus, also known as Human immunodeficiency virus), hepatitis B (Heparin-resistant hepatitis B virus), and influenza A/B virus, among others.
Furthermore, it offers a significant benefit from screening large amounts of high throughput specimens obtained from diverse sources and yields reproducible results, regardless of the complexity of the DNA template. Interestingly, PCR-sequencing tests can be carried out using standard PCR.
The difference between these methods is that the probe-binding sites are the target sequences (or other specific regions of interest), whereas the target fragments are the target sequences themselves. Also, each probe-binding site is unique because there are a finite number of such sites in the target sequences. Therefore, this strategy can provide good sensitivity for detecting specific fragments and small variations because the amount of sequence overlap is proportional to the total number of binding sites.
Primers are known as the basic instrument of DNA synthesis because they give rise to the amplified product. Primers are small sequences of DNA (the product of a PCR), which carry information regarding a target sequence, which is the targeted molecule or RNA molecule. Primers are usually labeled with a dye, such as fluorine (fluorine is a yellow color, e.g., in red and green dyes). Most primers contain a sequence of primers (also called ‘in situ primers). However, the most commonly amplified species-specific primers are designed specifically for the detection of specific target fragments, rather than sequences for which they are known. In addition to this, additional information regarding the selected target has to be provided during the development of primer sets, to enable the selection of the optimal set of primers according to this information. Although the amplified products are usually labeled with a dye, it can also be done with unbound fluorescein-diluted primers.
Specificity of primers, such as detection of insertion/deletion/ deletion events, insertion/deletion events or insertion/ insertion/ deletion events has been reported in various papers. Specificity of primers has been tested for the detection and/or discrimination of insertion/ deletion events, insertion/ deletion events and insertion/ insertion/ deletion events. Specificity of primers has also been tested as follows: to confirm insertion/ deletion events, insertion/ insertion-deletion events, insertion/ deletion events and insertion/ deletion events that do not involve insertion/ deletion events. Specificity of primers has also been tested for target detection of the following molecules: proteins, DNA from viruses (primers are used on both long and short fragments of DNA, as shown in, DNA from viral elements such as DNA from genes (primers are used on both long and short fragments of DNA.
PCR-Sequencing is based on the principle of using sequences from one species as templates for amplification. With PCR-Sequencing, PCR products can be sequenced using either direct or indirect probes. Direct probe detection can be carried out using labeled probes that hybridize directly to the target sequences, while indirect probe detection can be carried out by labeling the primers (using complementary probes), so as to produce binding sites, and the probe then hybridizes with the labeled primers at a later stage of the process.