| Business Data Model Data Model |
| Description | Genotyping assay techniques vary from single genomic regions that are interrogated using PCR reactions to high throughput assays examining genome-wide sequence and structural variation. The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by Kary Mullis [1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. [2] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. [3] The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. [4] As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. [5] Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. [6] The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions. |
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| Attribute Details |
PCR Annealation Temp
| Description | The annealation temperature at which the primers (custom-made, short DNA strands, specifically designed to bond to sites at the beginning and end of the segment to be copied) bind to the DNA. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Temperature [FLOAT(15)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Annealation Temperature
| Description | Polymerase Chain Reaction (PCR) Annealation Temperature. The temperature that is required to anneal or bind DNA strands. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Enumeration [VARCHAR(20)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Annealation Tm
| Description | The annealation time taken at which the primers (custom-made, short DNA strands, specifically designed to bond to sites at the beginning and end of the segment to be copied) bind to the DNA. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Time [TIME] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Cycle Number Qty
| Description | The number of PCR cycles needed to obtain the required strands of DNA. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Count [INTEGER] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Denaturation Temp
| Description | The temperature at which the hydrogen bonds are broken in the double stranded DNA, creating single strands of DNA that are susceptible to copying. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Temperature [FLOAT(15)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Denaturation Temperature
| Description | Polymerase Chain Reaction (PCR) Denaturation Temperature. The temperature that is required to break the double helix of DNA to single strands. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Enumeration [VARCHAR(20)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Denaturation Tm
| Description | The time taken for the hydrogen bonds to be broken in the double stranded DNA, creating single strands of DNA that are susceptible to copying. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Time [TIME] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Descr
| Description | The description of the PCR experiment. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Description Long [VARCHAR(1024)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Elongation Temp
| Description | The Taq polymerase enzyme adds DNA nucleotides from 5'to 3', reading the template from 3' to 5' side, making two double stranded molecules from each one double stranded DNA molecule that was denatured. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Temperature [FLOAT(15)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Elongation Temperature
| Description | Polymerase Chain Reaction (PCR) Elongation Temperature. The units of the temperature required to elongate the DNA. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Enumeration [VARCHAR(20)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Elongation Tm
| Description | The time taken for Taq polymerase enzyme to add DNA nucleotides from 5' to 3', reading the template from 3' to 5' side, making two double stranded molecules from each one double stranded DNA molecule that was denatured. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Time [TIME] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Forward Primer Txt
| Description | The forward primer extends in PCR from the start codon towards the stop codon of template DNA. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Text Medium [VARCHAR(255)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Forward Sequence Txt
| Description | Sequence of a strand in the 5-3 direction. For the forward strand, this means reading left-to-right. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Text Large [VARCHAR(1024)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Product Length Qty
| Description | Length of the Amplified DNA. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Decimal Float [FLOAT(15)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Reverse Primer Txt
| Description | The reverse primer extends from the stop codon towards the start codon |
| Data Type | Standards - Data Domains.ddm/Data Domains/Text Medium [VARCHAR(255)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Reverse Sequence Txt
| Description | Sequence of a strand in the 5-3 direction. For the reverse strand it means right-to-left. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Text Large [VARCHAR(1024)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Technician Txt
| Description | The name of the technician who performed the DNA amplification. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Text Small [VARCHAR(50)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
PCR Type
| Description | Polymerase Chain Reaction Type. 1) Assembly Polymerase Chain Reaction (PCR) or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product. 2) Helicase-dependent amplification: similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation. 3) Hot start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (for example, 95°C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature. 4) Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence. 5) Methylation-specific PCR (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation. 6) Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR. 7) Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by gel electrophoresis. 8) Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences. 9) Overlap-extension PCR or Splicing by overlap extension (SOE) : a genetic engineering technique that is used to splice together two or more DNA fragments that contain complementary sequences. It is used to join DNA pieces containing genes, regulatory sequences, or mutations; the technique enables creation of specific and long DNA constructs. 10) Quantitative PCR (Q-PCR): used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR (or QF-PCR) methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR. 11) Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends). 12) Thermal asymmetric interlaced PCR. 13) Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles. |
| Data Type | Standards - Data Domains.ddm/Data Domains/Enumeration [VARCHAR(20)] |
| Is Part Of PrimaryKey | false |
| Is Required | false |
| Is Derived | false |
| Is Surrogate Key | false |
| Generalization Details |
Generalization1
| Supertype | Assay |
| Subtype | PCR |
| Transform As | TABLE |
| Business Data Model Data Model |