Source : Molecular Cytogenetics Techniques and Applications
Operation method
basic program
Principle
Microdissected or flow-sorted chromosomes amplified by PCR to prepare DNA of the entire chromosome are considered the preferred route for high-quality probes that coat all of a given chromosome. This is the case for coating all 24 chromosomes in single chromosome, multicolor FISH (M-FISH), or spectral karyotyping. The procedure provided below is a modified version of the most original procedure for concatenated oligonucleotide-primed PCR (DOP-PCR) and is suitable for the amplification of chromosomal DNA with either SpectrumOrange™ dUTP or SpectrumGreen™ dUTP. The procedure can be used, with or without minor modifications, for the amplification of chromosomal DNA with a variety of other labeled dUTPs. DNA.The reaction involves an oligonucleotide with an Xho I restriction endonuclease cleavage site at the 5' end, a defined oligohexanucleotide sequence at the 3' end and a series of abbreviated nucleotide sequences in the middle (a random mixture of all tetranucleotides). Theoretical calculations indicate that there is one such defined hexanucleotide sequence every 4kb throughout the genome. Under the right conditions, amplification using this sequence as a primer can bypass the specific 3' sequence, perhaps by annealing one or more of the condensed nucleotides to improve stability, with the specific sequence at the 5' end allowing the primer to anneal at a higher temperature. In practice, the DOP-PCR procedure begins with several cycles of low annealing temperature (low-fidelity PCR), followed by multiple cycles of high annealing temperature (high-fidelity PCR), producing a mixture of randomly amplified DNA. Flow-sorted chromosomes are generally labeled by two rounds of amplification by DOP-PCR, with the products undergoing a third round of amplification in the presence of fluorescently labeled nucleotide triphosphate. Although the notch panning method can also be used to label probes for chromosome analysis, this step is generally not required to obtain high-quality probes.
Materials and Instruments
Flow cytometer sorted chromosomes (500 ~ 1000 chromosomes per PCR reaction) Move In general, flow-sorted chromosomes were resuspended and amplified in PCR analysis buffer under the following PCR conditions: 95°C, 5 min, followed by 9 cycles of 94°C, 1 min, 30°C, 1.5 min, and 72°C, 3 min, with a 2-min, 5-s ramp-up and down time; followed by 35 cycles of 94°C, 15 s, 62°C, 15 s, and 72°C, 15s (see Notes 8~10); then a separate 72°C extension was performed for 10min, and finally stored at 4°C. The above PCR products were re-amplified in 100μL of the system following the same PCR conditions. 10μL of amplified product was electrophoresed on a 1% agarose gel to form fragmented bands of 300~1000bp. The final yield of DNA ranged from 1.5 to 3.0μg. 〜The above PCR products were labeled in PCR labeling buffer using the PCR conditions for amplified DNA. 1 to 21 μL of labeled DNA could be used for hybridization without purification. Caveat 1. Contamination is a very serious problem in PCR operations. To avoid contamination of the PCR reaction, try to use the best quality reagents, which are used only for PCR. all solutions should preferably be dispensed and stored frozen at -20°C for later use. Also, pipette with aerosol-proof tips to minimize cross-contamination. Designate an area of the laboratory to be used exclusively for PCR operations. If possible, DNA sample preparation is performed in a separate laboratory. In addition, it is important to select an appropriate negative control (no target sequence) for each PCR operation. 2. PCR of specific target sequences requires titration of Taq enzyme. Too much enzyme can lead to non-specific background. 3. Vary the number of cycles to determine the appropriate number of cycles for the best signal-to-noise ratio. 4. The presence of large molecular masses of DNA (visible on electrophoresis as fragmented bands ranging in size from gel holes to approximately 2.0 kb) indicates that the labeled probe is inappropriate. Such probes can lead to non-specific, non-cellular components of the high background, which can sometimes be reduced by sonication. Determine the appropriate number of PCR cycles by titrating the Taq enzyme as mentioned in Note 9. For more product details, please visit Aladdin Scientific website.
10×PCR buffer PCR analysis solution PCR labeling buffer Primers Taq polymerase
PCR instrument