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Biotechnology - Mid > Lec8 | Genomic and Transcriptomic Analysis Techniques > Flashcards

Lec8 | Genomic and Transcriptomic Analysis Techniques Flashcards

1
Q

What cellular role does mRNA serve?

A

mRNA serves as an intermediary molecule, carrying genetic information from the nucleus to the cytoplasm for protein synthesis.

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2
Q

What happens when genes are in their active state?

A

When genes are in their active state, many copies of mRNA corresponding to those genes are produced.

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3
Q

What process is indirectly assessed by examining various mRNAs?

A

Examining various mRNAs allows for the indirect assessment of genetic information or gene expression.

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4
Q

Why is mRNA considered a surrogate marker?

A

mRNA is considered a surrogate marker because its presence and quantity indicate gene activity.

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5
Q

Why is mRNA converted to cDNA?

A

mRNA is converted to cDNA because mRNA is easily degraded, whereas cDNA is a more stable form.

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6
Q

What cellular processes do mRNA molecules directly facilitate?

A

mRNA molecules facilitate the processes of transcription in the cell nucleus and translation in the cytoplasm.

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7
Q

What is a DNA microarray used for?

A

A DNA microarray is used to analyze the expression of thousands of genes simultaneously.

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8
Q

How do DNA microarrays contribute to the understanding of diseases such as cancer?

A

DNA microarrays allow researchers to investigate gene activity within a cell or tissue sample, providing insights into gene regulation, gene function, and how genes are involved in disease development.

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9
Q

What does global expression profiling achieve?

A

Global expression profiling allows for the examination of gene expression without the influence of gene preselection.

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10
Q

How are different DNA sequences arranged on a microarray chip?

A

Different DNA fragments are arranged in rows and columns on a microarray chip, with the identity of each fragment known through its location.

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11
Q

What role does hydrogen bonding play in the function of a DNA microarray?

A

In a DNA microarray, hydrogen bonding facilitates complementary base pairing between the sample cDNA and the DNA probes on the array.

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12
Q

What factors influence the signal produced in a microarray experiment?

A

The signal in a microarray experiment depends on hybridization conditions, such as temperature, and the amount of target cDNA.

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13
Q

In a typical DNA microarray experiment, where do the mRNA samples come from?

A

In a typical DNA microarray experiment, the mRNA samples are collected from a reference sample, representing a healthy individual, and an experimental sample.

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14
Q

What is the next step in the DNA microarray experiment after mRNA is collected and before the microarray slide is used?

A

After collection of mRNA, the next step is to convert it to cDNA and label each sample with a different colored fluorescent probe.

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15
Q

What process follows the mixing of labeled cDNA samples?

A

After the labeled cDNA samples are mixed, they are allowed to bind to the microarray slide through a process called hybridization.

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16
Q

How is the expression of each gene measured on the microarray slide?

A

The microarray is scanned with a laser to measure the expression of each gene printed on the slide; the laser excites the fluorescence of the bound cDNA.

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17
Q

In a microarray, what information does a green spot yield when comparing a normal cell sample to a cancer cell sample?

A

In a microarray, a green spot indicates that a gene is expressed in normal cells but not in cancer cells.

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18
Q

If a red spot is present on a microarray, what does it signify about gene expression levels when comparing a normal cell sample to a cancer cell sample?

A

A red spot on a microarray signifies that a gene is expressed in cancer cells but not in normal cells.

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19
Q

What does a yellow spot indicate in a microarray experiment analyzing cancerous and normal cells?

A

A yellow spot on a microarray indicates that the gene is expressed in both normal and cancer cells.

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20
Q

What information does a black spot convey in a microarray experiment comparing cancerous and normal cells?

A

A black spot in a microarray experiment conveys that the gene is not expressed in either normal or cancer cells.

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21
Q

How can a DNA microarray be used in drug discovery regarding disease pathways?

A

DNA microarrays can be used to generate hypotheses for complex disease mechanisms and identify potential drug targets and pathways.

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22
Q

In the context of compound screening, what is one of the main uses of DNA microarrays?

A

In compound screening, DNA microarrays are used to characterize lead compounds for selectivity and specificity, as well as to identify molecules that disrupt the expression of intended disease genes.

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23
Q

What is a major limitation of DNA microarray technology?

A

A significant limitation of DNA microarray technology is that the results can be complex to interpret and are not always quantitative or reproducible.

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24
Q

If a patient’s gene expression changes from a red spot to a green spot after 6 weeks of treatment for breast cancer, is the treatment affecting the patient’s gene expression?

A

The treatment is affecting the patient’s gene expression by suppressing the gene’s expression.

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25
Q

If a patient’s gene expression changes from a red spot to a green spot after 6 weeks of treatment for breast cancer, is the treatment working?

A

Yes, the treatment may be working, as a reduction in the expression of the gene involved in causing cancer indicates that it could be inhibiting tumor growth.

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26
Q

What is DNA sequencing?

A

DNA sequencing is the process of determining the order of nucleotides—adenine (A), thymine (T), cytosine (C), and guanine (G)—along a DNA strand.

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27
Q

What are the general categories of DNA sequencing methodologies?

A

The two general categories of DNA sequencing methodologies are DNA sequencing by synthesis and single-molecule DNA sequencing.

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28
Q

How many DNA bases can be sequenced per reaction using Sanger DNA sequencing?

A

Sanger DNA sequencing can sequence 500 to 700 DNA bases per reaction.

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29
Q

How many reactions are performed per gel in Sanger sequencing?

A

In Sanger sequencing, 16 reactions are performed per gel.

30
Q

What is the range of DNA bases that can be sequenced per reaction using massively parallel DNA sequencing?

A

Massively parallel DNA sequencing can sequence 100 to 5,000 DNA bases per reaction.

31
Q

How many reactions per slide are possible with massively parallel DNA sequencing?

A

With massively parallel DNA sequencing, it is possible to have 10 thousand to 10 billion reactions per slide.

32
Q

What is the capacity of DNA bases that can be sequenced per pore in nanopore DNA sequencing?

A

Nanopore DNA sequencing has the capacity to sequence 10 thousand to 4 million DNA bases per pore.

33
Q

What is the range of pores per device in nanopore sequencing?

A

In nanopore sequencing, the range is from 40,000 to 250,000 pores per device.

34
Q

What is the underlying method behind Sanger’s chain termination sequencing?

A

Sanger’s chain termination sequencing is a PCR-based method.

35
Q

What type of reaction is employed in Sanger sequencing?

A

A modified DNA replication reaction involving both dNTPs and ddNTPs is employed in Sanger sequencing.

36
Q

How many reaction tubes are prepared in Sanger sequencing?

A

Four separate reaction tubes are prepared in Sanger sequencing.

37
Q

What are the common components present in each reaction tube in Sanger sequencing?

A

Each reaction tube contains identical DNA of interest, known as the template, primers, dNTPs, and DNA polymerase.

38
Q

What additional component is present in the Sanger sequencing reaction tubes, and why is it important?

A

Each reaction tube also contains a small amount of a specific ddNTP, which is used to terminate the chain at a known base.

39
Q

Why does the absence of a 3’-OH group in ddNTPs cause DNA synthesis to halt in Sanger sequencing?

A

The 3’-OH group is necessary for the formation of the phosphodiester bond; its absence in ddNTPs prevents further chain elongation, thus halting synthesis.

40
Q

How does the Sanger sequencing process achieve the separation of DNA strands for sequencing?

A

In the Sanger sequencing process, DNA strands are separated, typically through denaturation by heat.

41
Q

To which end of the DNA fragment does the radioactive primer bind during Sanger sequencing?

A

The radioactive primer binds to the 3’ end of the DNA fragment during Sanger sequencing.

42
Q

What does DNA polymerase synthesize in the Sanger method?

A

DNA polymerase synthesizes a complementary DNA sequence in the Sanger method.

43
Q

What is the result of using a specific ddNTP in the complementary strand during Sanger sequencing?

A

The use of a specific ddNTP in the complementary strand halts DNA synthesis at the base corresponding to that ddNTP.

44
Q

What is created as a result of chain termination at different positions in Sanger sequencing?

A

Chain termination at different positions results in the creation of DNA fragments of varying lengths.

45
Q

After generating different sized fragments in Sanger sequencing, how are they processed to determine the sequence?

A

The fragments generated in Sanger sequencing are separated with a gel to create a sequencing ladder that indicates the sequence.

46
Q

If ddATP is added to the “A tube,” what are the contents of the “A tube”?

A

The “A tube” contains all four dNTPs, DNA polymerase, primer, and ddATP.

47
Q

If you only use a forward primer in a Sanger sequencing reaction, what strand do you use as a template?

A

When using only a forward primer, the 3’ to 5’ strand should be used as the template.

48
Q

What is one of the disadvantages of Sanger sequencing concerning the length of sequences it can handle?

A

One disadvantage of Sanger sequencing is that it can only sequence 200 to 500 nucleotides in a single reaction.

49
Q

What are some advantages of Sanger sequencing?

A

Sanger sequencing is ideal for sequencing single genes, amplicons up to 100 base pairs, 96 samples or less, identifying microbes, analyzing fragments, and analyzing short tandem repeats.

50
Q

In Sanger sequencing, why does sequence quality degrade after 700 to 900 bases?

A

Sequence quality degrades after 700 to 900 bases in Sanger sequencing due to the limitations of the polymerase processivity and the depletion of reaction components.

51
Q

What is the underlying principle of Next-Generation Sequencing (NGS)?

A

The underlying principle of NGS is similar to Sanger sequencing, in that it relies on capillary electrophoresis.

52
Q

In NGS, how are the bases in each genomic fragment identified?

A

The bases in each genomic fragment are identified by emitted signals when the fragments are ligated against a template strand.

53
Q

What type of sequencing methodology does NGS utilize?

A

NGS utilizes array-based sequencing to process millions of reactions in parallel, resulting in very high speed and throughput at a reduced cost.

54
Q

What are the four major steps in the NGS process?

A

The four major steps in the NGS process are library preparation, amplification, sequencing, and analysis.

55
Q

How is the DNA prepared for sequencing in the NGS library preparation step?

A

In the library preparation step, DNA is fragmented, either enzymatically or by sonication, to create smaller strands.

56
Q

What role do adaptors play in NGS library preparation?

A

Adaptors, which are short, double-stranded pieces of synthetic DNA, are ligated to the DNA fragments with the help of DNA ligase.

57
Q

What are the three main sections of an adaptor used in NGS?

A

The three main sections of an NGS adaptor are the sequencer binding site, the index (barcode), and the sequencing primer binding site.

58
Q

What is the function of the sequencer binding site on the adaptor?

A

The sequencer binding site allows fragments of DNA to bind to the surface of the chip or beads in the sequencer.

59
Q

What does the index (barcode) region of the NGS adaptor do?

A

The index (barcode) region of the adaptor identifies which sample is being sequenced when multiple samples are processed at once.

60
Q

What occurs during the sequencing primer binding site region of the adaptor?

A

During sequencing, the primer binds to the primer binding site, allowing the polymerase to bind and extend during the sequencing reaction.

61
Q

What methods are used to amplify template DNA in NGS?

A

Template DNA is amplified via droplet or bridge PCR to generate millions of template molecules for the sequencing reaction.

62
Q

How is the signal generated during the sequencing step of NGS?

A

During the sequencing step, each nucleotide position on the template molecule generates an optical or chemical signal in response to processes like nucleotide addition.

63
Q

What happens to the signal generated during the sequencing step of NGS?

A

The signal generated during the sequencing step of NGS is recorded and then analyzed using bioinformatic tools to determine the DNA sequence.

64
Q

After the sequencing data is analyzed, how is the sequence assembled (NGS)?

A

The sequence is assembled by aligning overlapping sequence reads to reconstruct the original sequence, providing information about coverage and identity.

65
Q

How does third-generation sequencing (3rd GS) differ from previous sequencing technologies?

A

Unlike previous sequencing technologies that relied on short reads, 3rd GS generates long reads that can span entire genomic regions, providing a more complete picture of the genome.

66
Q

What specific technologies are encompassed by third-generation sequencing?

A

Third-generation sequencing encompasses technologies such as single-molecule real-time sequencing (SMRT) and nanopore sequencing.

67
Q

In SMRT sequencing, what is a single DNA molecule immobilized on?

A

In SMRT sequencing, a single DNA molecule is immobilized on a polymerase enzyme.

68
Q

How does SMRT sequencing read the DNA sequence in real-time?

A

In SMRT sequencing, polymerase incorporates fluorescently labeled nucleotides as it reads the DNA sequence in real-time.

69
Q

How long can the continuous reads generated by SMRT sequencing be?

A

The continuous reads generated by SMRT sequencing can be several kilobases in length.

70
Q

How does nanopore sequencing measure changes to read DNA sequence?

A

Nanopore sequencing works by threading DNA through a tiny pore and measuring changes in electrical conductivity as the DNA bases pass through.

71
Q

What is one major advantage of using third-generation sequencing technologies?

A

One major advantage is the ability to generate long reads that can span entire genomic regions, aiding in the study of complex areas.

72
Q

What are some disadvantages of third-generation sequencing technologies?

A

Disadvantages include a high error rate of approximately 15%, difficulty calling variants in complex regions, and the complexity of data analysis.

Biotechnology - Mid (11 decks)

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