What is a molecular beacon probe?
Molecular beacon probess are qPCR probes that contain a 5′ dye and 3′ quencher, and are designed to form a stem-loop (hairpin) structure. The loop contains sequences that hybridize to the target DNA and is flanked by complementary arms (typically 5–7 bp long) that form the stem, when the arms hybridize to each other.
What is the advantage of molecular beacons over TaqMan probe?
In contrast to the TaqMan probes described above, molecular beacons are not degraded during the amplification process but rather remain intact and must bind to the target in every cycle in order to produce measurable fluorescence.
What are molecular beacons used for?
Molecular beacons are structured probes that are highly sensitive, sequence specific, and are used for sequence detection in qPCR and in vitro studies.
What are sloppy molecular beacons?
Sloppy molecular beacons possess relatively long probe sequences, enabling them to form hybrids with amplicons from many different species despite the presence of mismatched base pairs.
What is a scorpion probe?
Scorpions® probes are highly sensitive, sequence-specific, bi-labeled fluorescent probe/primer hybrids designed for qPCR.
How do you make a molecular beacon?
Target Design
- The source and of the template and the sequence of the primers and template.
- Design primer pairs that amplify a target region of 75 to 250 basepairs.
- Avoid selecting a molecular beacon target sequence that forms strong secondary.
- Analyze the selected sequences using a DNA folding program such as DNA mfold.
How molecular beacons are used for real time detection and quantification of PCR products?
The molecular beacons are the ideal hybridization-based probe for short oligonucleotide detection, and, thus, it is a suitable probe for real-time PCR [11]. In real-time PCR, MB hybridizes with template DNA at the annealing step and produces the fluorescent signal directly.
How molecular beacon probe can be used to determine the genotype of an individual at a particular locus?
If a molecular beacon is designed to match a wild-type allele and another to match a mutant of the allele, the two can be used to identify the genotype of an individual. If only the first probe’s fluorophore wavelength is detected during the assay then the individual is homozygous to the wild type.
What are hydrolysis probes?
Hydrolysis probes are a popular detection chemistry for monitoring sequence-specific amplification in RTPCR. Just like with SYBR Green dye, signal detection is achieved through monitoring an increase in fluorescence as the reaction proceeds.
What is SYBR Green and how does it work?
SYBR Green I is a dsDNA binding dye, which can be used to quantify amplicon amount during the course of the PCR by tracking overall fluorescence emission. The dye binds into the minor groove of dsDNA, and does not bind to ssDNA. When bound, it increases its fluorescence by up to 100 fold (Figure 6).
Why is it called TaqMan probe?
TaqMan probes were named after the videogame Pac-Man (Taq Polymerase + PacMan = TaqMan) as its mechanism is based on the Pac-Man principle.
How does a TaqMan probe function?
The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection.
What is SYBR Green probe?
SYBR Green–based detection TaqMan-based detection. Uses Applied Biosystems™ SYBR™ Green dye (a dsDNA binding dye) to detect PCR product as it accumulates during PCR. Uses a fluorogenic probe specific to target gene to detect target as it accumulates during PCR.
Do molecular beacons fluoresce upon hybridization?
Molecular beacons: probes that fluoresce upon hybridization Nat Biotechnol. 1996 Mar;14(3):303-8.doi: 10.1038/nbt0396-303. Authors S Tyagi 1 , F R Kramer Affiliation 1Department of Molecular Genetics, Public Health Research Institute, New York, NY 10016, USA. [email protected]
What are molecular beacons?
Molecular beacons: probes that fluoresce upon hybridization Nat Biotechnol. 1996 Mar;14(3):303-8.doi: 10.1038/nbt0396-303.
What are nucleic acid probes?
We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fIuorogenic conforma-tional change when they hybridize to their targets.
What happens when DNA probes hybridize to their targets?
These probes undergo a spontaneous fIuorogenic conforma-tional change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion.