7 theme: PCR Sequencing

Question 1

What is pyrosequencing used for?

Answer 1

Suitable for bacterial genomes, not for big genomes, suitable for identification of non-cultivable microorganisms.

Question 2

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.

Answer 2

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

Question 3

What is pyrosequencing technique based on?

Answer 3

based on the detection of released pyrophosphate (PPi) during nucleotide incorporation.

Question 4

What is SOLiD?

Answer 4

Sequencing by Oligonucleotide Ligation and Detection

Question 5

What is the technology of SOLiD?

Answer 5

sequences DNA through repeated rounds of ligation of short, fluorescently labeled oligonucleotides.

Question 6

What is giving the colour of the signal in SOLiD sequencing and how it is detected?

Answer 6

Fluorescent probe; by digital camera.

Question 7

(SOLiD) The colour, signal of the fluorescent probe depends on ….., meaning there is…….different possibilities of the colour

Answer 7

..2 first bases of oligonucleotide
….4

Question 8

(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.

Answer 8

…The probe binds.
…DNA ligase enzyme
…enzyme
3
Fluorescence probe

Question 9

(SOLiD)The decoding is done by analyzing the …..

Answer 9

Overlapping nucleotides.

Question 10

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.

Answer 10

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.

Question 11

SMRT sequencing meaning

Answer 11

Single Molecule Real-Time Sequencing

Question 12

What is ZMW? What does it mean?

Answer 12

Zero Mode Waveguide; the presence of electromagnetic energy in a small volume near the pin hole (from SMRT)

Question 13

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).

Answer 13

fluorescent colour
zmw

Question 14

What is digital PCR used for?

Answer 14

to quantify nucleic acids (such as DNA or RNA) at very low concentrations. Also for detecting rare mutations.

Question 15

Positive signal in digital PCR means…

Answer 15

the presence of at least one target molecule

Question 16

Droplet digital PCR. How can we determine which droplets contain target DNA/RNA and which do not?

Answer 16

by increased fluorescensed intensity

Question 17

How methylation turns off genes?

Answer 17

When methyl groups are added to cytosine bases in DNA. That leads to gene silencing and reduced gene expression.

Question 18

What does methylation maintenance mean?

Answer 18

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.

Question 19

How is the primary enzyme responsible for methylation maintenance called?

Answer 19

DNA methyltransferase 1 (DNMT1).

Question 20

How does methylated bases affect genome?

Answer 20

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.

Question 21

What does LAMP mean?

Answer 21

Loop mediated isothermal amplification

Question 22

What is FLOS-LAMP?

Answer 22

It is a LAMP technique, that incorporates fluorescence detection to monitor the amplification process in real-time.

Question 23

What are used to introduce the mutation into the gene?

Answer 23

Oligonucleotides with desired mutations

Question 24

What type of mutations oligonucleotides can introduce?

Answer 24

point mutations (base-pair substitution), insertion mutation or deletion into the gene sequence.

Question 25

Mutagenesis. Deletion oligonucleotide is used for what?

Answer 25

To delete the fragment of cloned DNA and create a desired mutation in plasmid.