Fluorescent Techniques in PCR. Cao jian ping. PCR. Amplification of specific gene or gene fragments Process of PCR 1. Denaturation 2. Annealing 3. Extension. Traditional way to assay the result of PCR:
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Fluorescent Techniques in PCR
Cao jian ping
Agarose gel electrophoresis and subsequent staining with ethidium bromide
Detect through the binding of double-strand DNA specific dyes, these kinds of dyes have essentially no fluorescence of their own and become intensely fluorescent when they bind to nucleic acids.
Include an additional hybridization to an internal part of the amplified product and detect the PCR product by generating a signal change only when the correct product is amplified
The left tube contains non-complementary single-stranded oligonucleotide and the right tube contains complementary single-stranded. The sample concentration in left tube is five times as that in right tube. The were photographed using ASA 400 film.
Nicke Svanvik,* Gunnar Westman,† Dongyuan Wang,* and Mikael Kubista*,1.(1999)
It is due to electron donating ability, which permits the charge transfer between the nucleobase and a nearby dye.
Irina Nazarenko, Rick Pires, Brian Lowe, Mohamad Obaidy and Ayoub Rashtchian. (2002)
The oligonucleotide labeled close to the 3’ end showed substantial quenching upon hybridization, while the oligonucleotide labeled close to the 5’ end exhibited no decrease in fluorescence.
Fluoreophore as donor, quencher as acceptor
1. The transfer efficiency is in proportion to the inverse sixth power of the distance between two emission groups.
2. Effective range: 20-100 A
Salvatore A. E. Marras*, Fred Russell Kramer and Sanjay Tyagi(2002)
Salvatore A. E. Marras*, Fred Russell Kramer and Sanjay Tyagi
An oligonucleotide probe is labeled at one end with a fluorophore and in the middle or at the other end with a fluorescence quencher. When the probe binds to the target site in a PCR product, the 5’–3’exonuclease activity of Taq DNA polymerase cleaves it freeing the fluorophore from quencher, thereby producing an increase in fluorescence.
1. Optimal distance: 6 to 14 bases. (Ju et al., 1995, 1996)
2. Location of the quencher from the traditional 3’ position to an internal one increases the sensitivity of probe.
Sheering without cleavage is detrimental for the assay.
Molecular beacons are single-stranded nucleic acid molecules with a stem-and-loop structure. The loop portion of the molecular beacon hairpin is complementary to a target sequence. The stem is formed by two sequences that are complementary to each other. In the hairpin formation, the fluorophore, attached to one end of the probe, is quenched by the quencher, attached to the other end of the probe, and no fluorescence is emitted. On hybridization of the molecular beacon to its target the stem dissociates, causing the quencher and the fluorophore to move away from each other, resulting in the restoration of fluorescence.
Structure and principle of operation of molecular beacons.
Musa M. Mhlanga*,† and Lovisa Malmberg‡,1.(2001)
Phase transitions of molecular beacons.When annealed, the molecular beacon–target hybrid is fluorescent. As the temperature is raised, the probe–target hybrid denatures and a nonfluorescent molecular beacon in a closed formation is formed. On further rise in temperature, the hairpin stem denatures and the molecular beacon forms a fluorescent random coil.
“window of discrimination.”: higher fluorescence perfectly complementary hybrids can be distinguished from mismatched hybrids
Bottom curve: in the absence of target. Top curve:in the presence of perfectly complementary target. Middle curve:in the presence of a target containing a mismatched nucleotide at the same position.
In the same oligonucleotide the quencher remains close enough to partly quench the fluorophore by a non-collisional mechanism
1.A PCR primer with a hairpin structure at the 5’-extension
2. The linker stops DNA polymerase from replicating the stem-loop.
3. When primer extends and the target is synthesized, the stem-loop unfolds and hybridizes with target
4. Unimolecular mechanism ensures faster kinetics and greater stability of the probe-target complex
combination of the molecular beacon with DNAzymes
1. Deoxyribose-phosphate backbone of DNA has been replaced by a structurally homomorphous backbone of N- (2-aminoethyl) glycine units in PNA.
2. PNA is charge-neutral
3. It hybridizes faster, and forms more stable sequence than the natural nucleic acids
4. PNA/DNA complex is either double or triple-stranded
1. Strongly fluorescent upon hybridization
2. Fold back onto the PNA interacting with its bases, causing free-probe fluorescence
A single-base mismatch is sufficient to prevent probe hybridization and subsequent fluorescence enhancement.
Bind at annealing temperature and dissociate during elongation
a) Chemical structure of light-up probes.
b) Light-up probes interact with single-stranded target nucleic acid.
Nicke Svanvik,* Gunnar Westman,† Dongyuan Wang,* and Mikael Kubista*,1.(1999)
The free probe fluorescence may vary significantly, since the dye interacts differently with four nucleobases.
The fluorescence enhancement upon hybridization of is mainly determined by the free probe fluorescence.
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