Science
Exploring the Principles of Probing in QPCR in 2023
Probe-based quantitative PCR (qPCR) probe is an extension of non-quantitative PCR technology in which specific DNA ‘probe’ sequences replace the PCR primers.
It allows for measuring gene expression levels by measuring Tm (melting temperature) using a thermal cycler. The principle behind probing in QPCR is that it uses fluorescently labelled primers and probes to detect the presence of a specific sequence and then amplifies the signal using a thermostable DNA polymerase.
Here are some key principles of probe-based qPCR;
Fluorescent Probe
The principle of the probe in QPCR is that the fluorescent probes have a sequence complementary to the target sequence, and the probe can be bound to the target DNA by complementary base pairing. The fluorescence quenching effect is caused by the non-specific binding of the probe to non-target DNA strands, which increases background fluorescence and decreases target gene amplification efficiency. Therefore, it is necessary to select appropriate fluorescent probes with high specificity for target genes to not interfere with non-specific binding.
Probe Hybridization
Hybridization is the reaction of two complementary single-stranded DNA or RNA molecules to form a double-stranded molecule. In QPCR, hybridization is used to detect the presence of target DNA/RNA. The probe is labelled with a fluorescent dye that emits light when excited by a laser. The fluorescence emitted from the probe will be captured by a CCD camera and analyzed by software. A threshold can be set to determine if there is enough signal fluorescence.
Quantification
Quantification is the most important principle of probes in QPCR. The probe will be designed to have a fluorescent reporter and a quencher. Usually, the reporter is attached to the 5′ end of the probe, and the quencher is attached to its 3′ end. When these two groups are close together, they can form a base pair called an oligonucleotide duplex (OD). In this situation, it would be impossible for the reporter to fluoresce because fluorescence requires a certain amount of energy which would be absorbed by the quencher group instead. However, when no DNA is present in the reaction mixture, no OD will be formed so that energy can pass through and cause fluorescence emission.
Fluorescence detection
QPCR probes are fluorescently labelled oligonucleotides that bind directly to the target sequence. QPCR probes are designed to be complementary to a segment of a target gene. The polymerase chain reaction (PCR) is then used to amplify the fragment of interest, which allows for the detection of the amplified fragment by the probe. The probe hybridizes with its target sequence and emits fluorescence when excited at the appropriate wavelength, allowing real-time detection.
Probe cleavage
Probes are designed to be cleaved by the target DNA or RNA. The cleavage reaction is catalyzed by a thermostable endonuclease (Taq polymerase). In some cases, two probes are used, one each for the target and reference gene. The second probe is a control to ensure the reaction works properly. The principle of probe cleavage is based on a sequence-specific interaction between the probe and its target DNA or RNA. This interaction is usually between a part of the probe sequence and a complementary base pair in the target molecule.
Conclusion
The probe is the key component of QPCR. The probe should be bound to the target DNA (or RNA) with a specific base sequence. Such a probe will then trigger a fluorescence reporter attached to the QPCR machine and can be detected during PCR amplification. This process can quantitatively measure the target DNA (or RNA) amount under certain conditions.
NEW YORK — To save power, NASA turned off another scientific equipment on its long-running Voyager 2 spacecraft.
NASA Switches Off Instrument On the Spacecraft To Save Power
The space agency announced on Tuesday that 2’s plasma science instrument, meant to study the movement of charged atoms, was turned off in late September to allow the spacecraft to continue exploring for as long as possible, which is estimated to be into the 2030s.
NASA turned off a suite of instruments on Voyager 2 and its twin, Voyager 1, after exploring the gas giant planets in the 1980s. Both are currently in interstellar space or the region between stars. The plasma instrument on Voyager 1 stopped working years ago and was finally shut off in 2007.
The remaining four instruments on 2 will continue to collect data on magnetic fields and particles. Its mission is to investigate the regions of space beyond the sun’s protective sphere.
NASA Switches Off Instrument On Voyager 2 Spacecraft To Save Power
It launched in 1977, is the only spacecraft to have visited Uranus and Neptune. It is now more than 12 billion miles (19.31 billion kilometers) from Earth. 1 is more than 15 billion miles (24.14 billion kilometers) beyond Earth.
SOURCE | AP
MIAMI — Hurricane Kirk’s waves could generate life-threatening surf and rip current conditions this weekend throughout the United States East Coast, as well as in Bermuda, the Greater Antilles, and the Bahamas, according to forecasters.
Kirk was a Category 3 hurricane in the middle Atlantic Ocean that might grow further but was predicted to stay away from land, according to the Miami-based National Hurricane Center on Thursday.
Hurricane Kirk Could Cause Dangerous Surf Conditions Along The US East Coast
Kirk-generated swells were forecast to reach parts of the Leeward Islands on Friday, Bermuda and the Greater Antilles on Saturday, and the East Coast and the Bahamas on Sunday, according to the center.
No coastal watches or warnings were in effect. The major storm was around 1,130 miles (1,820 kilometers) east of the Leeward Islands, with maximum sustained winds of 125 mph (205 km/h).
Meanwhile, Tropical Storm Leslie formed late Wednesday in the eastern Atlantic and is expected to strengthen into a hurricane in the following days, forecasters said. It was also not considered a threat to the land.
Hurricane Kirk Could Cause Dangerous Surf Conditions Along The US East Coast
The storm was about 540 miles (870 kilometers) southwest of Cabo Verde’s southernmost tip, with maximum sustained winds of 45 mph (75 kph), according to the center.
The storms raged in the Atlantic as rescuers in the United States Southeast sought for missing persons after Hurricane Helene struck last week, leaving a trail of death and devastation.
SOURCE | AP
NASA has announced astronauts Butch Wilmore and Suni Williams are safely in orbit on the first crewed flight test of Boeing’s Starliner spacecraft heading for the International Space Station.
As part of NASA’s Boeing Crew Flight Test, the astronauts launched a ULA (United Launch Alliance) Atlas V rocket from Space Launch Complex-41 at Cape Canaveral Space Force Station in Florida at 10:52 a.m. EDT Wednesday for an end-to-end test of the Starliner system.
“Two brave NASA astronauts are well on their way to this historic first test flight of a brand-new spacecraft,” stated NASA Administrator Bill Nelson. “Boeing’s Starliner represents a new era of American exploration. Human spaceflight is a risky endeavor, but it is worth it. It is an exciting time for NASA, our commercial partners, and the future of space exploration. “Go Starliner, Butch, and Suni!”
The flight test is part of NASA’s Commercial Crew Program and will help validate the transportation system, launch pad, rocket, spacecraft, in-orbit operations capabilities, and return to Earth with astronauts aboard as the agency prepares to certify Starliner for rotational missions to the space station. Starliner has already completed two uncrewed orbital missions, including a test to and from the space station, as well as a pad abort demonstration.
Boeing Starliner Makes Orbit
“With Starliner’s launch, separation from the rocket, and arrival in orbit, Boeing’s Crew Flight Test is right on track,” said Mark Nappi, vice president and program manager for Boeing’s Commercial Crew Program. “Everyone is focused on giving Suni and Butch a safe, comfortable, ride and performing a successful test mission from start to finish.”
Boeing’s mission control center in Houston will supervise a sequence of autonomous spacecraft maneuvers while Starliner is in flight. NASA teams will supervise space station activities from the Mission Control Center at the agency’s Johnson Space Center in Houston.
“Flying crew on Starliner represents over a decade of work by the Commercial Crew Program and our partners at Boeing and ULA,” said Steve Stich, Commercial Crew Program Manager at NASA’s Johnson Space Center in Houston. “For many of us, this is a career-defining occasion, ushering in a new crew transportation capacity for our agency and our country. We will take it one step at a time, putting Starliner through its paces and remaining watchful until Butch and Suni safely land back on Earth at the end of this test journey.”
At about 12:15 p.m., Starliner will dock autonomously to the forward-facing port of the station’s Harmony module. Thursday, June 6, and will remain at the orbital laboratory for almost a week.
Wilmore and Williams will help ensure that the spacecraft is functioning properly by testing the environmental control system, the displays and control system, and moving the thrusters, among other things, during flight.
Wilmore and Williams will join the Expedition 71 crew, which includes NASA astronauts Michael Barratt, Matt Dominick, Tracy C. Dyson, and Jeanette Epps, as well as Roscosmos cosmonauts Nikolai Chub, Alexander Grebenkin, and Oleg Kononenko.
NASA’s arrival and in-flight event coverage is as follows (all times Eastern and subject to change depending on real-time operations):
NASA Television channels will continue to broadcast the Starliner’s mission.
Thursday, June 6
9:30 a.m. – Arrival coverage begins on NASA+, the NASA app, and YouTube, and continues on NASA Television and the agency’s website.
12:15 p.m. – Targeted docking
2 p.m. – Hatch opening
2:20 p.m. – Welcome remarks
3:30 p.m. – Post-docking news conference at NASA Johnson with the following participants:
- NASA Associate Administrator Jim Free
- Steve Stich, manager, NASA’s Commercial Crew Program
- Jeff Arend, manager for systems engineering and integration, NASA’s International Space Station Office
- Mark Nappi, vice president and program manager, Commercial Crew Program, Boeing
Coverage of the post-docking news conference will air live on NASA+, NASA Television, the NASA app, YouTube, and the agency’s website.
To attend the post-docking briefing, U.S. media must contact the NASA Johnson newsroom at: [email protected] or 281-483-5111 by 1 p.m. Thursday, June 6. To join by phone, media must contact the NASA Johnson newsroom by 3 p.m. Thursday, June 6.
5:50 p.m. – NASA Administrator Bill Nelson, Deputy Administrator Pam Melroy, Associate Administrator Jim Free, Associate Administrator for Space Operations Ken Bowersox, and Johnson Space Center Director Vanessa Wyche will speak with Wilmore and Williams about their launch aboard the Starliner spacecraft.
Coverage of the Earth to space call will air live on NASA+, NASA Television, the NASA app, YouTube, and the agency’s website.
Saturday, June 8
8:50 a.m. – NASA astronauts Wilmore and Williams will provide a tour of Starliner.
Coverage of the in-orbit event will stream live on NASA+, NASA Television, the NASA app, YouTube, and the agency’s website.
Monday, June 10
11 a.m. – Williams will speak to students from Sunita L. Williams Elementary School in Needham, Massachusetts, in an event aboard the space station.
Coverage of the Earth to space call will air live on NASA+, NASA Television, the NASA app, YouTube, and the agency’s website.
Tuesday, June 11
3:15 p.m. – Wilmore will speak to students from Tennessee Tech University in an event aboard the space station.
Coverage of the Earth to space call will air live on NASA+, NASA Television, the NASA app, YouTube, and the agency’s website.
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