7 theme: PCR Sequencing Flashcards
What is pyrosequencing used for?
Suitable for bacterial genomes, not for big genomes, suitable for identification of non-cultivable microorganisms.
Pyrosequencing process:
1) DNA Preparation: The DNA to be sequenced is first ……………. to create single strands, which are then immobilized and primed for sequencing.
2) Nucleotide Addition: One type of nucleotide (A, T, C, or G) is added to the reaction mixture at a time.
3) Detection of Pyrophosphate:
If the added nucleotide is ………….. to the next base on the template DNA strand, it is incorporated by ……………
…………of the nucleotide releases a ………………..
If the nucleotide does not match the template base, it is not incorporated, and no ……….
4) Enzyme Cascade and Light Emission: The released pyrophosphate triggers a series of enzyme-catalyzed reactions:
…… converts pyrophosphate into ….
…….uses this ATP to convert luciferin to ………., producing light.
!!!The amount of light generated is proportional to the …….., indicating how many nucleotides were ……..
5) Detection and Data Collection: The emitted light is detected by a camera, creating a series of light signals (peaks), known as a ………. Each peak represents a specific ………… incorporated into the growing DNA strand, allowing researchers to deduce the sequence.
1) denatured
3) complementary
by DNA polymerase.
Incorporation
a pyrophosphate (PPi) molecule.
no pyrophosphate is released.
4)
Sulfurylase ; ATP.
Luciferase ; oxyluciferin
amount of pyrophosphate released, incorporated.
5) pyrogram; specific nucleotide
What is pyrosequencing technique based on?
based on the detection of released pyrophosphate (PPi) during nucleotide incorporation.
What is SOLiD?
Sequencing by Oligonucleotide Ligation and Detection
What is the technology of SOLiD?
sequences DNA through repeated rounds of ligation of short, fluorescently labeled oligonucleotides.
What is giving the colour of the signal in SOLiD sequencing and how it is detected?
Fluorescent probe; by digital camera.
(SOLiD) The colour, signal of the fluorescent probe depends on ….., meaning there is…….different possibilities of the colour
..2 first bases of oligonucleotide
….4
(SOLiD) If the first two nucleotides form base paring, then……..Once the probe binds, we use…… to ligate probe with a primer. The information (the given colour) is saved by digital camera. Then, using an …… the last…..nucleorides are cleaved. As a result, the………is removed. We continue until n-4 primer is done.
…The probe binds.
…DNA ligase enzyme
…enzyme
3
Fluorescence probe
(SOLiD)The decoding is done by analyzing the …..
Overlapping nucleotides.
Illumina Solexa sequencing . The process:
1) After …. DNA, we generate smaller fragments
2) All fragments are ….. with DNA …..on …… ends
3) ds DNA is …….
4) Binding to a chip: ……
5) ……..sequence is synthesized. It’s done for all the fragments.
6) The single strand, which is not attached to the flow cells oligonucleotides gets ……
7)…..
8) ……synthesizes complementary sequence
9) DNA is …….., both fragments stick to the plate. The process repeats (bridge building)
10) result: …… and….. strands. …….strands are cleaved.
11) Nucleotides are ……….labeled. When nucleotide joins the complementary sequence, it then will be …. by a laser, and the fluorescent …… is obtained.
12) by …. we can now generate the whole genome.
1) isolating
2) ligated ; adaptors
both
3) denaturated
4) hybridization
5) complementary
6) washed away
7) bridge building
8) polymerase
9) denatured
10) reverse and forward; reverse
11) fluorescently; excited; signal.
12) overlay.
SMRT sequencing meaning
Single Molecule Real-Time Sequencing
What is ZMW? What does it mean?
Zero Mode Waveguide; the presence of electromagnetic energy in a small volume near the pin hole (from SMRT)
SMRT: Each nucleotide is labeled with …….; ONLY THOSE NUCEOTIDES, THAT COME UNDER ……… GIVES FLUORESCENCE (because its only this region, that has ability to excite the fluorescent molecule).
fluorescent colour
zmw
What is digital PCR used for?
to quantify nucleic acids (such as DNA or RNA) at very low concentrations. Also for detecting rare mutations.
Positive signal in digital PCR means…
the presence of at least one target molecule
Droplet digital PCR. How can we determine which droplets contain target DNA/RNA and which do not?
by increased fluorescensed intensity
How methylation turns off genes?
When methyl groups are added to cytosine bases in DNA. That leads to gene silencing and reduced gene expression.
What does methylation maintenance mean?
It refers to the process by which DNA methylation patterns are preserved during DNA replication, ensuring that methylation is faithfully passed from parent cells to daughter cells.
How is the primary enzyme responsible for methylation maintenance called?
DNA methyltransferase 1 (DNMT1).
How does methylated bases affect genome?
X-Chromosome Inactivation (DNA methylation plays a key role in inactivating one X chromosome by methylating genes across the chromosome, ensuring that only one X chromosome is transcriptionally active.)
Proper methylation is essential for healthy development and function, while abnormal methylation patterns can lead to a wide range of diseases.
DNA methylation causes genetic imprinting= certain genes are expressed in a monoallelic manner, meaning only one allele (either maternal or paternal) is expressed, while the other is silenced.
What does LAMP mean?
Loop mediated isothermal amplification
What is FLOS-LAMP?
It is a LAMP technique, that incorporates fluorescence detection to monitor the amplification process in real-time.
What are used to introduce the mutation into the gene?
Oligonucleotides with desired mutations
What type of mutations oligonucleotides can introduce?
point mutations (base-pair substitution), insertion mutation or deletion into the gene sequence.
Mutagenesis. Deletion oligonucleotide is used for what?
To delete the fragment of cloned DNA and create a desired mutation in plasmid.
