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MCM Test 2 > Molecular Diagnostics > Flashcards

Molecular Diagnostics Flashcards

1
Q

Hybridization and
PCR

are used for what?

A

They can be used to

  1. Identify infectious agents: however the sequence of the pathogen must be known.
  2. Identify a inherited disorder: however the sequence of the gene must be known.
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2
Q

What must we know before performing hybridization and PCR?

A

The sequence of pathogen and gene

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3
Q

How many pathogens have been sequenced?

A

The sequence of 5,000 pathogens have been sequenced

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4
Q

Flies and humans share ____ of genes

A

50%

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5
Q

Hybridization ideology

A

ssDNA has a high propensity to bind with another strand of DNA/RNA that is complimentary.

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6
Q

When do we use hybridization?

A

Hybridization is used to detect a particular DNA/RNA sequence in a mixture of other DNA/RNA.

Short, single stranded oligonucleotides (probes) are designed and radioactively labeled and then immobilized on a a membrane (blotting). The goal is for them to behind with a target nucleic acid that has a complimentary sequence.

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7
Q

_______ is a technique that is based on hybridization.

A

Blotting.

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8
Q

Types of blotting

A

Southern
Northern
Western

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9
Q

Southern blotting

A

Both the probe and target nucleic acids are DNA

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10
Q

Northern blotting

A

Probe is an antibody

Target is RNA

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11
Q

Western blotting

A

Probe is a ssDNA

Target is protein

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12
Q

How does blotting occur?

A

Say you have a cup with a long strand of DNA. You want to see if it contains gene A, your gene of interest.

  1. Take the DNA and cleave it, resulting in smaller pieces.
  2. Run a gel electrophoresis to seperate the DNA fragments based on size and charge
  3. Transfer the gel onto a membrane (blot), which will also transfer the fragments.
  4. Expose the membrane to our radioactive DNA probe, which is the compliment to the gene of interest. If our membrane has our gene of interest, the probe will hybridize with it
  5. Use a Xray to visualize it.
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13
Q

Purpose of southern blot

A

Determine which restriction fragments are associated with a gene of interest.

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14
Q

Purpose of northern blot

A

Measure the [size] and [quantity] of mRNA

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15
Q

Purpose of western blot

A

Measure the [amount] of protein or antibody.

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16
Q

What is the purpose of Polymerase Chain Reaction (PCR)?

A

The purpose of PCR is to amplify a sample of DNA to create millions of copies

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17
Q

How is PCR conducted?

A
  1. Select a region of dsDNA that you want to amplify
  2. Heat to separate strands
  3. Cool to anneal primers
  4. Add [Taq polymerase] and all 4 dNTPS (deoxynucleotide triphosphates)
  5. Amount of DNA is now doubled.
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18
Q

How are the primers designed in PCR?

A

Primers are designed to flank each end of DNA in a 3’–>5’ direction.

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19
Q

What is Taq polymerase?

A

creates the copy of DNA by extending the primers.

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20
Q

Where is PCR conducted in?

A

Thermocycler

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21
Q

Advantage of PCR

A

We only need a small amount of template DNA: there is a 10^9 fold amplification

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22
Q

Disadvantage of PCR

A
  1. You need to know the sequence of the flanking DNA for the primers
  2. errors
  3. you can amplify contaminated DNA
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23
Q

Why is PCR ran?

A

PCR is ran so that you can amplify DNA. Many machines require you to have a certain amount of DNA or you may need more than 1 copy to manipulate the DNA.

Thus, you can use it to

  1. Detect infectious agents
  2. Detect genetic mutations
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24
Q

1 cycle of PCR makes ____
2 cycle makes ____
3 cycles makes ____

A

2
4
8

2^n

n=trial

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25
Q

is PCR quantitative?

A

No. We are making millions of copies

So we have invented a new method: qPCR

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26
Q

If we were asked:

What method would we perform if we were asked to quantitatively measure much how our gene expression changes in cancer?

A

qPCR

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27
Q

What does qPCR do?

And how is it different than PCR?

A

qPCR is used to quantify the copy number of a specific gene.

The only difference between PCR and qPCR is that a probe is used which fluoresces only in the presence of the PCR

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28
Q

What is the probe in a qPCR

A

It is usually a complimentary oligo with a fluorescent tag.

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29
Q

qPCR is used to do what?

A
  1. Detect the LEVELS of an infectious agent

2. Determine LEVELS of gene expression

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30
Q

In the graph indicating [fluorescence vs. cycle numbers], what does a low Ct amount mean?

A

low Ct= high amounts of targeted nucleic acids

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31
Q

What does a high Ct amount mean?

A

High Ct= lower amounts of targeted nucleic acids.

Thus, more amount of cycle numbers were needed to produce the same amount of target sequence than the one with the low Ct. In other words, it has less of the target sequence that you’re looking for.

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32
Q

_____ is often used to come up with more specific answers

A

qPCR

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33
Q

What 2 properties of our DNA can we use to use PCR and hybridization to answer specific questions?

A
  1. RFLP–> restriction fragment length polymorphism

2. VNTR–> variable number of tandem repeats

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34
Q

RFLP and VNTR can be used for

A
  1. Forensics

2. Dianostics such as (prenatal diagnosis, newborn screenings and genetic carriers)

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35
Q

RFLP Theory and How It is Done

A

Peoples genome differs by 1 in every 1000 BP. Some of these differences are in the recognition sequence for restriction enzymes. Thus, when attacked by a restriction enzyme, different fragments are made.

In RFLP,

  1. The genomes of 3 individuals (1 from evidence and 2 from suspects) are cleaved with the same set of restriction enzymes.
  2. Fragments are then loaded onto a agarose gel, producing different patterns.
  3. DNA is then transferred to a NYLON membrane
  4. Conduct Southern Blot
    a. Add DNA probe and wash off excess.
    b. Put in under Xray

The person of interest will have fragments of the same length, because the same restriction enzyme was used.

36
Q

RFLP is used in

A
  1. Forensic analysis,
  2. DNA fingerprinting,
  3. paternity testing
  4. inheritance of a dz
37
Q

If we wanted to run a RFLP and our sample was limited, what could we do?

A

Run a PCR before

38
Q

2 parents want to use their DNA to determine if their daughter will have sickle cell anemia, what test is ran?

A

RFLP

RFLP can be used to detect mutations

39
Q

How can RFLP be used to detect for mutations?

A

When a person has a mutation, often times the site where restriction enzymes act is altered. You can thus under RFLP using the same restriction enzymes to see if your DNA is cleaved how the mutated DNA would be cleaved like.

40
Q

How can RFLP be used to detect for sickle cell?

A

In a normal HB gene, the restriction enzyme 3Ddel will cut at three different places.

Patients with sickle cell, however will only have 2 restriction sites.

Knowing this information, you can run a RFLP to detect for mutations.

41
Q

Variable number of tandem repeats (VNTR) is used for?

A

VNTR is used to identify diseases and indicate the severity

42
Q

What is VNTR

A

Patterns of short tandem repeats (short repeats of the same nucleotide) occur in the genome, but vary in individuals

43
Q

What can VNTR be used to detect

A
  1. Huntingtons

2. Fragile X syndrome

44
Q

People with Huntingtons have a large number of _____ copies

A

CAG

45
Q

We can make a large scale production of recombinant proteins such as

A
  1. insulin
  2. eyrthropoietin
  3. growth factors
  4. clotting factors
  5. vaccines
46
Q

How do we create insulin?

A

We need a: plasmid vector and peice of cDNA.

cDNA is inserted in a plasmid vector to produce a recombinant DNA.

Recombinant DNA is then inserted into a bacteria.

Using a fermentation tank, the recombinant DNA will then produce our insulin, which is then extracted from the bacteria.

47
Q

How is the flu vaccine made?

A

FDA selects 3 strains of vaccines it thinks may be selected.

Strains are then incubated in a chicken egg and mixed and distributed.

48
Q

Flu vaccine starts in ______ and vaccinination begins in ______

A

January

October

49
Q

The production of antibodies/antigens are becmoing very common.

A

ANTIBODIES

50
Q

What do antibodies do?

A

Antibodies are released from your body to attack foreign aliens.

51
Q

Abciximab

A

a monoclonal antibody that inhibits the aggregation of platelets

52
Q

Basiliximab

A

a monoclonal antibody that prevents the rejection of a kidney transplant

53
Q

Cetuximab

A

a monoclonal antibody that treats colorectal cancer

54
Q

Infliximab

A

a monoclonal antibody that treats autoimmune diseases

55
Q

Retuximab

A

a monoclonal antibody that treats lymphomas and leukemia

56
Q

how are monoclonal antibodies made?

A
  1. Rat in injected with antigen
  2. Spleen cells of the rat will begin to produce antibodies.
  3. Fuse spleen cells with tumor cells, creating hybridomas and causing it to become immortilized.
  4. Culture in a HAT medium and select for + cells (cells that you want to bind to the epitope of interest.
  5. Harvest the monoclonal cells
57
Q

Monoclonal antibodies have been used to try to treat alzheimers. why hasnt this worked?

A

Researchers are thinking that the researchers are selecting the wrong epitope for the antibody to bind to

58
Q

Antibodies are also called

A

B lymphocytes

59
Q

ELISA stands for what?

A

Enzyme-linked immunosorbent assay

60
Q

What does ELISA do?

A

ELISA will test for the CONCENTRATION of antigens or antibodies in a sample

61
Q

Sandwhich ELISA

A

Measuring the amount of antigen in serum

Thus, the well is coated with antibodies

62
Q

Indirect ELISA

A

Measure the amount of antibodies in serum

Thus, the well is coated with antigen

63
Q

In sandwich ELISA, we are testing for the amount of _______ in the sample

A

antiGIN

64
Q

How do we run an elisa

A
  1. Well is coated with an immobilized antibody
  2. A sample with antigens is added. If the specific antigen is present, it will bind to the antibody.
  3. Wash excess antigens
  4. Add a a second antigen+HRP (Horserasdish peroxidase), which will bind to the specific antigen, if present.
  5. Wash
  6. A substrate (tetramethylbenzidine) is added and the enzyme will cause a color change.
65
Q

What does the color change in an ELISA tell us?

A

The rate of the color change tells us the amount of antigen/antibody that is present.

66
Q

What enzyme is used in ELISA to initiate color change?

A

HRP: Horseradish peroxidase

67
Q

What substrate is added to an indirect ELISA?

A

TMB: tetramethylbenzidine

68
Q

How do we test for HIV?

A

HIV testing is performed using indirect ELISA.

Wells are coated with HIV antigens.
We load the well with our serum. If we have HIV antibodies, they will bind to the antigen and we will see this.

69
Q

+ of ELISA

A

Efficient,
Sensitive
Cheap

70
Q
  • of ELISA
A

Can produce false positives and false-negatives

SOOOO confirm with western blot

71
Q

MI can be detected by performing what?

A

Sandwhich ELISA.

During an MI, we have elevated levels of Tropinin I and Tropinin T (both antigens).

Thus, we perform a sandwhich ELISA to test for levels of antigen (tropinin I and tropinin T)

72
Q

What do pregnancy tests for

A

HCG

73
Q

Is HCG an antibody or antigen?

A

Antigen

74
Q

What kind of test is a pregnancy test?

A

Sandwich ELISA

We are looking for levels of HCG (an antigen) in our urine.

75
Q

Pregnancy test has 3 sites

A
  1. Reaction site (Site where you pee on)
  2. Test site (site that tells you results)
  3. Control site (proves test is working)
76
Q

Reaction site of a pregnancy test

A

On the reaction site of a pregnancy test:

  1. Free HCG antibodies are located.
  2. We pee on the stick and if we have HCG antigens, it will bind.
  3. migrates to the test site
77
Q

Test site of pregnancy test

A
  1. has immobolized HCG antibodies
  2. When antigens+ab complex comes in, it will bind to the immobilized HCG antibodies.
  3. dye will turn colors.
78
Q

Western blotting allows you to

A

VISUALIZE your results.

79
Q

Western blot is also called

A

immunoblotting

80
Q

Western blotting is used for what?

A

to detect levels of proteins

Our proteins are always antigens, so we are ALWAYS taking the antigen

81
Q

Western blotting test

A
  1. Run our sample of proteins through a SDS page, which will separate our proteins (antigens) based on charge and size (smaller will travel faster).
  2. Transfer to a nitrocellulose membrane
  3. Add primary AB
  4. Add a secondary AB with a tag. Tag will turn the fragmented line colors!

Thicker the line, more of the target protein present.

82
Q

What tests do we do to CONFIRM HIV?

A

Western blot

83
Q

In qPCR, a sample that has a higher amount of target DNA would reach the threshold at a earlier/later cycle?

A

Earlier cycle

lower Ct

84
Q

An increase in the amount of PCR product depends on what?

A

the amount of target sequence being amplified.

85
Q

_______________ are being made into popular drugs.

A

monoclonal antibodies.

86
Q

Why are monoclonal antibodies called what they are?

A

They are specific for a single epitope on a antigen.

MCM Test 2 (12 decks)

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