07 Dot Blot Assay

Question 1

It is also known as enteric fever, is a communicable disease, found more likely in man and occurs due to systemic infection mainly by Salmonella typhi organisms, a gram-negative bacterium.

Answer 1

● Typhoid fever

Question 2

Typhoid fever, also known as enteric fever, is a communicable disease, found more likely in man and occurs due to systemic infection mainly by _____ typhi organisms, a ____ bacterium

Answer 2

Salmonella

gram-negative

Question 3

Environmental exposure to Salmonella typhi may be more frequent in ____, presumably due to sex-linked differences in hygiene practices and dining-out behavior. But, it predominantly affects the __________

Answer 3

males

children of school-age or younger.

Question 4

These diseases are spread through sewage contamination of food or water and through person-to-person contact. People who are currently ill and people who have recovered but are still passing the bacteria in their feces can spread ________. Thus, it is through the _______

Answer 4

Salmonella Typhi

fecal–oral route.

Question 5

It is an acute generalized infection of the reticuloendothelial system, intestinal lymphoid tissue, and the gallbladder.

Answer 5

Typhoid fever

Question 6

It is a potentially fatal multisystemic illness that causes nearly 220,000 deaths annually and 22 million illnesses per year.

Answer 6

Typhoid fever

Question 7

Isolation of Salmonella from ________ is the most reliable means of confirming an infection. Blood culture is regarded as the gold standard for diagnosis and carry 70-75% diagnostic yield in the first week of illness

Answer 7

blood, urine or stool

Question 8

______can be more quickly analyzed using dot blots or slot blots.

Answer 8

DNA and RNA

Question 9

DNA and RNA can be more quickly analyzed using dot blots or slot blots. These procedures are applied to:
(3)

Answer 9

• Expression
• Mutation
• Amplification/deletion analyses

Question 10

Techniques in which target or probe sequences are immobilized on a solid matrix.

Answer 10

Blots

Question 11

It follows a similar principle to Western or Southern blotting, except that the sample is not blotted from a gel. Instead, samples are dotted directly onto a membrane before being probed for detection.

Answer 11

Dot Blot Technique

Question 12

multiple samples are immobilized in a geometric array on a nitrocellulose or nylon membrane

Answer 12

Dot Blot Principle

Question 13

______ is a method for detecting biological samples like proteins or nucleic acids.

Answer 13

Dot blot

Question 14

Samples are dotted directly on to a membrane before being probed for detection.

Question 15

The target DNA or RNA is deposited ____ on the membrane by means of various devices, some with vacuum systems.

Answer 15

directly

Question 16

A _____can be used for procedures testing only a few samples.

Answer 16

pipet

Question 17

For _____, the target is deposited in a circle or dot.

Answer 17

dot blots

Question 18

_____ are useful for multiple qualitative analyses where many targets are being compared, such as in mutational screening

Answer 18

Dot blots

Question 19

Performed most efficiently in less complex samples, such as cloned plasmids, PCR products, or selected mRNA preparations

Answer 19

dot blot

t

Question 20

It is important that the probe hybridization conditions be optimized because cross-hybridizations cannot be definitively distinguished from true target identification.

Question 21

A _______ serves as the baseline for interpretation of this dot blot assay.

Answer 21

negative control

Question 22

Applications of Dot Blot:

Answer 22

• Monitoring labeling efficiency
• Estimating protein concentration
• Comparing antibody performance

Question 23

Dot Blot Analysis:

Answer 23

• ImageJ

- UN-SCAN-IT gel Analysis Software

Question 24

_____ plugin used to measure microarray image stacks. Use the control panel that appears to define the ROI (circle or rectangle) grid.

Answer 24

ImageJ

Question 25

______ is an image analysis program extensively used in the biological sciences and beyond.

Answer 25

ImageJ

Question 26

There are two built-in methods for analyzing a dot blot in ImageJ:

Answer 26

1. To treat each row as a horizontal “lane” and use ImageJ’s gel analysis function.
2. To subtract the background and measure the integrated density of each dot.

Question 27

__________ turns your scanner into a gel densitometer and allows you to automatically analyze gel electrophoresis images.

Answer 27

UN-SCAN-IT gel Analysis Software

Question 28

Problems encountered using Dot Blot Method

Answer 28

● High background on the blot
● Weak or no signal from the blot
● Non-specific binding

Question 29

• often caused by too high concentration of the antibody, which can bind to PVDF membranes
• the buffers used may be too old
• film overexposed or became wet during exposure
• short blocking time or washing intensity

Answer 29

High background on the blot

Question 30

• can be caused by low concentration of antibody or antigen

- lack of protein and this can come from poor sample preparation

Answer 30

● Weak or no signal from the blot

Question 31

• Short blocking time can lead to non-specific binding by the primary and secondary antibodies
• Too much time in the blocking buffer can lead to poor binding

Answer 31

● Non-specific binding

Question 32

It is a test that relies on biochemistry to measure the presence and/or concentration of an analyte. The analyte can be large proteins, antibodies that a person has produced as a result of an infection or small molecules.

Answer 32

Immunoassay

Question 33

based on the agglutination reaction

characterized either by gel formation in a liquid phase or as an opaque band in an agar plate assay.

Answer 33

First immunoassay formats

Question 34

only occurs when the right amounts of antibody and antigen are present.

Answer 34

Agglutination

Question 35

Positive agglutination test –>

Answer 35

appears cloudy

Question 36

⮚ is characterized by a ‘button’ at the bottom of the reaction vessel that is formed by non-reacted particles.

⮚ may indicate the mismatch of antibody to antigen or it may be obtained from either excess antigen or excess antibody.

Answer 36

Negative result of agglutination test

Question 37

are commonly used for the detection of microbial pathogen antigens in blood serum as they provide rapid results with a minimum of equipment.

Answer 37

Agglutination immunoassays

Question 38

These are routinely used as diagnostic methods that detect antigens of infectious agents or antibodies against them in bodily fluid.

Answer 38

Enzyme linked immunosorbent assays (ELISAs)

Question 39

o is achieved either directly, or indirectly by the use of a coating antibody (also called capture antibody), which actively traps antigen in the solid phase.

Answer 39

Immobilization

Question 40

● Enzyme immunosorbent assays can be classified into:

Answer 40

o	 Direct (e.g. double antibody sandwich) 
o	Indirect (e.g. triple antibody sandwich) ELISAs