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Molecular Diagnostics > DISCRIMINATION TECHNIQUES > Flashcards

DISCRIMINATION TECHNIQUES Flashcards

1
Q

Explain the general categories of dicrimination techniques

A
  1. Electrophoretic separation
    ~Provides physical separation of individual nucleic acids according to molecular weight and shape
  2. Altenatives to electrophoresis
    ~Determines the size or sequence of nucleic acid without electrophoresis
  3. Hybridization assays
    ~Provides visualization of specific nucleic acids out of a background, usually by the use of probes.
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2
Q

Explain the principle of electrophoresis

A
  1. Both DNA and RNA are negatively charged and will migrate towards the anode when an electric field is present.
  2. Nucleic acids to be separated may be prepared in several ways before electrophoresis according to need.
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3
Q

What is electrophoretic separation and how does it aid in clinical assays?

A

Electrophoretic separation is a laboratory technique that uses an electric field to separate molecules based on their size and charge. In clinical assays, it helps identify and quantify specific DNA or protein molecules, providing critical information for diagnosing and monitoring diseases.

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4
Q

How are different nucleic acid molecule populations identified in gel electrophoresis?

A

Different populations of nucleic acid molecules are identified by their distinct banding patterns on the gel. Each band corresponds to a molecule of a specific size. By comparing the bands to a DNA ladder (marker), the sizes of the nucleic acids can be determined.

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4
Q

Describe the process of visualizing DNA fragments using a dye in gel electrophoresis

A

In gel electrophoresis, DNA fragments are separated in an agarose gel. A dye, such as ethidium bromide or SYBR Green, binds to the DNA. Under UV light, these dyes fluoresce, making the DNA fragments visible as bands on the gel.

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5
Q

Explain how the size of DNA fragments is determined using commercially available DNA markers.

A

DNA markers, also known as ladders, contain fragments of known lengths. By running these markers alongside the sample DNA, researchers can compare the bands formed by the sample to those of the marker. The migration distance of the bands indicates the size of the DNA fragments.

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6
Q

What role does molecular weight play in the separation of nucleic acid molecules during gel electrophoresis?

A

Molecular weight affects the migration speed of nucleic acid molecules through the gel. Smaller molecules move faster and travel further than larger ones. This differential migration allows for the separation and analysis of nucleic acids of different sizes.

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7
Q

Why is it important to use a dye in visualizing DNA fragments during gel electrophoresis?

A

Using a dye is important because it binds to the DNA and fluoresces under UV light, making the DNA fragments visible. Without the dye, the DNA would be invisible to the naked eye, making it impossible to analyze the results of the electrophoresis.

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8
Q

Discuss the significance of using DNA markers in gel electrophoresis.

A

DNA markers provide a reference for determining the size of sample DNA fragments. By comparing the sample bands to the known sizes of the marker bands, researchers can accurately measure the lengths of the DNA fragments in their samples.

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9
Q

ow does gel electrophoresis help in the diagnosis of genetic disorders?

A

Gel electrophoresis helps diagnose genetic disorders by separating and identifying DNA fragments associated with specific genes. By analyzing these fragments, clinicians can detect genetic mutations or abnormalities that cause diseases.

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9
Q

What are the common dyes used in visualizing DNA fragments in gel electrophoresis, and what are their properties

A

Common dyes include ethidium bromide and SYBR Green. Ethidium bromide intercalates with DNA and fluoresces under UV light. SYBR Green binds to DNA and RNA and also fluoresces under UV light. Both dyes are sensitive and provide clear visualization of nucleic acid fragments.

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10
Q

How can electrophoretic separation be used to interpret results in clinical assays, specifically in identifying different molecular weights?

A

Electrophoretic separation allows for the analysis of nucleic acid or protein fragments based on their size and charge. By comparing the separated bands to molecular weight markers, researchers can identify and quantify specific fragments. This information is crucial for interpreting clinical assays and making accurate diagnoses.

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10
Q

mention two types of polymers commoly used in electrophoresis

A
  1. agalose
  2. polyacrylamide
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11
Q

what determines the choice of polymer and polymer concentration

A
  1. The size of nucleic acid to be separated
  2. The resolution that is required
  3. How gels will be visualized.
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11
Q

What is the range of nucleic acid fragment sizes that agarose gel can separate?

A

Agarose gel can separate nucleic acid fragments ranging from as small as 20 base pairs (bp) to more than 10 megabases (Mb).

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12
Q

What is the typical resolution of separation in agarose gel?

A

The typical resolution of separation in agarose gel is limited to a size difference of 2% to 5%.

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12
Q

Are agarose gels permeable to fluorescent dyes?

A

Yes, agarose gels are permeable to fluorescent dyes.

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13
Q

ow can the results of an agarose gel electrophoresis be recorded?

A

The results can be recorded by a photographic image of the stained gel under UV light

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13
Q

What is the size difference range that agarose gel can usually resolve?

A

Agarose gel can usually resolve nucleic acid fragments with a size difference of 2% to 5%.

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14
Q

when are polyacrylamide polymers used

A

These are useful for high resolution separation of short molecules of up to 2kb

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15
Q

when are polyacrylamide polymers used primaliry

A

they are used for single stranded nucleic acid separation.

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15
Q

Factors affecting migration of Nucleic acids

A
  1. Size and complexity of nucleic acids
  2. Concentration of ethidium bromide
  3. Gel concentration
  4. Applied field
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16
Q

How does the size of nucleic acids affect their migration in gel electrophoresis?

A

The size of nucleic acids affects their migration such that smaller molecules travel faster through the gel than larger ones.

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17
Q

What role does DNA conformation play in the migration of DNA molecules during electrophoresis?

A

DNA conformation significantly affects the movement of DNA; for example, supercoiled DNA moves faster than relaxed DNA.

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18
Q

Why do smaller DNA molecules travel faster than larger ones in gel electrophoresis?

A

Smaller DNA molecules travel faster because they can move more easily through the pores of the gel matrix compared to larger molecules.

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19
Q

Explain how the complexity of nucleic acids influences their separation during electrophoresis.

A

The complexity of nucleic acids, such as whether they are linear, supercoiled, or relaxed, influences their separation because it affects how they move through the gel. More complex conformations like supercoiled DNA travel faster than simpler, relaxed conformations.

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20
Q

What is the effect of DNA supercoiling on its migration speed in gel electrophoresis compared to relaxed DNA?

A

Supercoiled DNA migrates faster than relaxed DNA because the coiled structure allows it to move more quickly through the gel matrix.

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21
Q

How does ethidium bromide affect the superhelicity of DNA molecules in gel electrophoresis?

A

Ethidium bromide intercalates into DNA and affects its superhelicity, thereby influencing its movement in the gel.

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22
Q

What happens to DNA molecules when the concentration of ethidium bromide is increased?

A

Increased concentration of ethidium bromide intercalated into DNA can change the DNA from a negatively supercoiled molecule to a fully relaxed form.

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22
Q

What is the effect of ethidium bromide on the migration speed of supercoiled versus relaxed DNA?

A

Supercoiled DNA, when intercalated with ethidium bromide, will move faster through the gel compared to relaxed DNA because the intercalation process changes the DNA’s conformation and affects its movement.

22
Q

Why is the concentration of ethidium bromide important in gel electrophoresis?

A

The concentration of ethidium bromide is important because it affects the superhelicity and consequently the movement of DNA molecules in the gel, influencing the results of electrophoresis.

22
Q

Describe the process by which ethidium bromide intercalates into DNA.

A

Ethidium bromide intercalates between the base pairs of the DNA molecule, inserting itself into the DNA structure and altering its superhelicity.

22
Q

How does gel concentration affect the pore size of the gel?

A

The gel concentration determines the pore size of the gel; the higher the gel concentration, the smaller the pores.

22
Q

What impact does increasing agarose gel concentration have on DNA migration and separation?

A

Increasing agarose gel concentration reduces the migration speed of DNA and improves the separation of smaller DNA molecules

22
Q

Why is gel concentration important in determining the resolution of DNA in gel electrophoresis?

A

The resolution of DNA changes with the percentage concentration of the gel; higher concentrations provide better resolution for smaller DNA molecules, while lower concentrations are better for separating larger DNA molecules.

22
Q

Explain the effect of lowering gel concentration on the separation of DNA molecules.

A

Lowering the gel concentration permits the separation of larger DNA molecules because the larger pores allow these bigger molecules to migrate more easily.

22
Q

How does the applied field affect the rate of DNA migration in gel electrophoresis?

A

The rate of migration of the DNA is proportional to the voltage applied. The higher the voltage, the faster the DNA moves.

22
Q

Describe how the pore size of the gel changes with varying concentrations of agarose.

A

The pore size of the gel becomes smaller with higher concentrations of agarose, and larger with lower concentrations. This change in pore size affects how DNA molecules of different sizes migrate through the gel.

22
Q

What happens to the migration speed of DNA molecules when the voltage is increased during electrophoresis?

A

When the voltage is increased, the migration speed of the DNA molecules also increases, causing them to move faster through the gel

22
Q

use of southern blot

A

Used widely in the detection of specific DNA fragments in DNA samples

22
Q

list 7 general steps in southern blot method

A
  1. Extract and purify DNA from cells
  2. DNA is restricted with enzymes
  3. Denature the DNA and electrophorese it. Denaturation separates the dsDNA into two ssDNA molecules
  4. Transfer to nitrocellulose paper
  5. Block with excess DNA: incubate with a ssDNA probe
  6. Wash off unbound probes
  7. Autoradiograph
22
Q

Why is it important to control the voltage applied during gel electrophoresis?

A

It is important to control the voltage to ensure consistent and accurate separation of DNA molecules. High voltage can lead to faster migration but may also cause the gel to overheat, potentially affecting the resolution and quality of the separation.

22
Q

Explain the relationship between voltage and DNA migration in electrophoresis.

A

There is a direct relationship between voltage and DNA migration; as the voltage increases, the rate at which DNA molecules move through the gel also increases.

22
Q

How can the rate of DNA migration be manipulated during an electrophoresis experiment?

A

The rate of DNA migration can be manipulated by adjusting the applied voltage. Increasing the voltage will speed up the migration, while decreasing the voltage will slow it down.

22
Q

Who developed the Northern blot technique, and in what year?

A

by James Alwine, David Kemp, and George Stark in 1977.

22
Q

metion 4 other southern blotting metods

A
  1. Northern blot
  2. Western blot
  3. Eastern blot 4.Southwestern blot
23
Q

What is the formal name for the Northern blot, and how are RNA molecules separated?

A

The formal name for the Northern blot is RNA blot.
RNA molecules are separated by gel electrophoresis, typically using an agarose gel.

23
Q

What type of nucleic acid does the Northern blot use for testing, and how does it differ from DNA?

A

The Northern blot uses RNA (including isolated mRNA) rather than DNA as the test nucleic acid. This differs from DNA because RNA is single-stranded and often includes mRNA, which is directly involved in protein synthesis.

24
Q

Why do RNA molecules appear as a smear rather than discrete bands on an agarose gel during Northern blotting?

A

RNA molecules appear as a smear rather than discrete bands because there are so many different RNA molecules of varying lengths present on the gel.

25
Q

To what material is RNA transferred after separation by gel electrophoresis in Northern blotting, and what are the alternatives?

A

After separation by gel electrophoresis, RNA is transferred to a nitrocellulose membrane. Alternatives include other types of paper or membranes.

25
Q

How does the probe bind to its target sequence in blotting techniques?

A

The probe forms base pairs with its complementary RNA sequence, resulting in a double-stranded RNA-DNA molecule.

25
Q

What type of molecule is used as a probe in blotting techniques, and what is its form?

A

The probe used in blotting techniques is single-stranded DNA.

26
Q

What are the common labels used on probes in blotting techniques, and why are they used?

A

Probes are commonly labeled with either radioactivity or an enzyme (such as alkaline phosphatase or horseradish peroxidase) to make them detectable.

27
Q

How is the location of the probe revealed when it has an enzyme bound to it?

A

The location of the probe is revealed by incubating it with a colorless substrate that the attached enzyme converts into a colored product that can be seen.

28
Q

How can the location of a probe labeled with radioactivity be detected in blotting techniques?

A

If the probe is labeled with radioactivity, it can expose X-ray film directly, revealing its location

29
Q

Northen blot allows us to observe a particular gene’s expression pattern between

A
  1. Tissues
  2. Organs
  3. Developmental stages
  4. Pathogen infection
30
Q

northen blot applications

A
  1. Observe particular gene expression rates in normal and abnormal or diseased conditions
  2. Observe the over expression of oncogenes and down regulation of tumor-supressor gene in cancerous cells as compared to normal tissue
  3. Observe gene expression in the rejection of transplanted organs
30
Q

altenatives to electrophoresis

A
  1. DNA Sequencing techniques
    a. Sanger dideoxy DNA b.sequencing method
    Pyrosequencing
  2. High-Performance Liquid Chromatography (HPLC)
  3. Mass spectroscopy
30
Q

On which strands is the DNA sequence analyzed, and why is this important?

A

The DNA sequence is analyzed on both strands (sense and antisense). This is important because it ensures accurate and comprehensive identification of genetic information by verifying the sequence on both complementary strands.

31
Q

What is the error rate when determining the DNA sequence and comparing it with a reference sequence?

A

The error rate when determining the DNA sequence and comparing it with a reference sequence is 0.1%.

31
Q

Why is it significant to identify deviations from the reference sequence in DNA analysis?

A

Identifying deviations from the reference sequence is significant because it allows for the detection of mutations or variations that may impact gene function, protein production, and can be associated with diseases or genetic disorders.

32
Q

How are deviations from the reference sequence identified?

A

Deviations from the reference sequence are identified using a computer program that compares the determined DNA sequence with the reference sequence.

32
Q

What types of base changes can be identified through DNA sequencing analysis

A

DNA sequencing analysis can identify base changes that result in an altered amino acid code, stop codons, deletions, or insertions

32
Q

what is the other name for sanger dideoxy DNA method

A

Also referred to as the chain termination DNA sequencing

33
Q

which principle is used in chain termination

A

Single stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another by polyacrylamide gel electrophoresis’.

34
Q

what is mass spectroscopy used for

A

to detect polymorphi

35
Q

what is (High-performance Liquid chromatography ) HPLC used for

A

HPLC is commonly used to separate and purify oligonucleotides

36
Q

explain prosequencing

A

Pyrosequencing allows the determination of nucleic acid sequence of short segments without the use of electrophoresis

37
Q

4 enzymes aded during pro sequesncing

A

DNA polymerase
ATP sulfurylase
Luciferase
Apyrase

38
Q

What reaction does DNA polymerase catalyze and what is released during prosequesncing process?

A

DNA polymerase catalyses the incorporation of complementary nucleotides, accompanied by release of pyrophosphate

38
Q

What is the relationship between the concentration of pyrophosphate released and the amount of incorporated nucleotides?

A

The concentration of pyrophosphate released is equimolar to the amount of incorporated nucleotides.

38
Q

How is the release of pyrophosphate monitored in this biochemical process?

A

The release of pyrophosphate is monitored by its conversion, along with adenosine-5’-phosphate, into ATP by the enzyme ATP sulfurylase.

38
Q

What subsequent reaction does ATP drive in the detection process, and what is the outcome?

A

ATP drives the conversion of luciferin into oxyluciferin, which produces visible light. This light production allows the detection and quantification of the incorporated nucleotides.

38
Q

What role does ATP sulfurylase play in the described biochemical process?

A

ATP sulfurylase converts pyrophosphate and adenosine-5’-phosphate into ATP, which is then used to convert luciferin into oxyluciferin, producing visible light.

Molecular Diagnostics (10 decks)

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