Molecular diagnostics

Question 1

Name 5 characteristics of a good test

Answer 1

1. sensitive
2. specific
3. high throughput
4. cheap
5. automation

Question 2

How does FISH work?

Answer 2

The target sequence is labeled, denatured with the probe. Where there is binding you will see hybridization of the probe to the target DNA which gives off a fluorescent signal

Question 3

For what can IVIS (intravital microscopy) be used?

Answer 3

To study drug behavior to help explain the different possible scenarios of a drug. e.g. localization, incomplete penetration

Question 4

How does FRET work? (fluorescent resonance energy transfer) and what can it be used for?

Answer 4

There is a donor and acceptor, when they overlap they interact and you’ll get excitation so there will be a fluorescent signal. This can be used to study protein-protein interaction

Question 5

How does competitive ELISA work?

Answer 5

There is an enzyme-couple antibody detector with fluorescent-labeled antibodies. If the enzyme binds then there is no competition, so you get a signal. But i the auto-antibody binds then you have 100% competition and no color emission so you can conclude whether a tumor antigen is present.

Question 6

How does agglutination assay work and when?

Answer 6

There is a carrier molecule that you incubate with your antibody e.g. anti-BCL2. If there is no BCL2 then the antibody will bind to the carrier and there will be clumping. But if BCL2 is present then there is no clumping.

This can be used to detect tumor antigens on protein level.

Question 7

What is a titer?

Answer 7

The maximum dilution at which visible agglutination is observed

Question 8

How does EMIT work? (enzyme multiplied immunoassay technique)

Answer 8

There is competition between the drug and a derivative conjugated enzyme. If the antibody binds to the conjugated derivative it will result in the inactivation of the enzyme as it blocks the active site. But if there is an antigen present, then the antibody will bind to the antigen and the enzyme will be active.

Question 9

How does AlphaLISA work? (amplified luminescent proximity homogeneous assay-linked immunosorbent assay)

Answer 9

There is a donor and acceptor. If the tumor antibody is present then there will be energy transfer that will emit a signal through oxygen channeling.

Question 10

How does luminex work?

Answer 10

In a 96-well plate there are beads labeled with a specific infra-red and are coated with an unique antibody that will bind to a certain bead. If there are 2 signals then there is presence of an antigen (reporter molecule + bead), if not then there will be only one signal

Question 11

Why is luminex a good technique?

Answer 11

Because it enables simultaneous detection and quantitation of multiple secreted proteins

Question 12

How does SNP analysis work?

Answer 12

There are probes that are designed to hybridize with the mutant. Two oligos will hybridize to the target sequence on the suspected mutant. Then ligation will take place and create one continuous strand to amplify (PCR) if there is a mutant present. If not, then there will be no ligation.

Question 13

How MLPA work?

Answer 13

The probe is built out of a primer binding sequence that is identical to each target and there are oligos that are specific to the target and a stuffer sequence which is a different length for each target. The probe will bind to the wild type and can be detected with capillary electrophoresis. So there is an absence of a probe when there is a mutant present.

Question 14

How does capillary electrophoresis work?

Answer 14

There is an electrical field present in a narrow tube where the samples can be added. Charged molecules will migrate through the tube, the smallest fragments migrate faster compared to the bigger ones.

Question 15

What are the advantages of capillary electrophoresis compared to gel electrophoresis?

Answer 15

There is less lateral diffusion and no temperature difference along the tube.

Question 16

Why is MPLA better than OLA?

Answer 16

Because you can add extra nucleotides to the epp so you can detect multiple mutations at once. So multifactorial diseases can be detected.

Question 17

Name 3 examples of what MLPA can detect

Answer 17

Methylation, dystrophin deletion, BRAF mutation in melanoma.

Question 18

Name 3 examples of what FISH can detect

Answer 18

Translocation, amplifications and deletions e.g. HER2 or ERG mutation

Question 19

How does array comparative genomic hybridization (CGH) work?

Answer 19

There are glass plates with a labeled DNA mix that are differentially labeled. The spots on the plate represent exons and the gDNA will hybridize which creates a signal to detect loss/gain of DNA.

Question 20

For what can array-CGH be used?

Answer 20

To detect large genomic profiles and changes e.g. insertions and deletions

Question 21

How does whole exome sequencing work?

Answer 21

The patient material is isolated and sheared into smaller fragments, then ligate adapters are added to each fragment and there will be bead capture of the exon fragments (exome capturing) which then can be sequenced and analysed.

Question 22

What is the advantage of WES compared to WGS?

Answer 22

WGS is very expensive and takes a lot of data analysis whereas WES only targets the protein-coding portion which harbors 85% of all known disease-causing mutations.

Question 23

How does NGS work? (bridge amplification)

Answer 23

There is the fragmentation of the DNA template so you go from gDNA to dsDNA by sonication and there is ligation of adapter molecules to the blunt need of the template DNA. Then immobilization happens of ssDNA to the flow cell, where the template will hybridize with the complementary probes on the surface and you get elongation of the strand. The original ssDNA is removed by denaturation and there is bridge formation by means of immobilization which synthesized a second strand to form a cluster of generated ssDNA. These are then sequenced, and the nucleotides are labeled with a different dye, by washing to get color imaging.

Question 24

How does FACS work in single cell sequencing?

Answer 24

FACS isolates highly purified single cells by tagging cells with a fluorescent marker protein

Question 25

How does LCM work in single cell sequencing?

Answer 25

LCM utilizes a laser system aided by a computer to isolate cells from solid samples

Question 26

What can single cell sequencing be used for?

Answer 26

To look for drug resistance due to heterogenous expression of the target, and to identify genes involved in stem cell regulation

Question 27

Name 4 methods that can be used to study RNA level diagnostics

Answer 27

RT-PCR, microarray, RNA sequencing, and miRNA profilling

Question 28

Name 5 methods that can be used for genetic testing for mutations

Answer 28

SNP analysis, MLPA, FISH, array-CGH, and whole exome sequencing

Question 29

Name 4 methods to study immunological diagnostics on protein level

Answer 29

Agglutination reaction, EMIT, AlphaLISA, and luminex

Question 30

Name a method that can be used in immunological diagnostics on antibody level

Answer 30

Competitive ELISA