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LBYBI13 > IMMUNOLOGIC AND MOLECULAR DIAGNOSTICS II > Flashcards

IMMUNOLOGIC AND MOLECULAR DIAGNOSTICS II Flashcards

1
Q

Molecular-based diagnostic methods are notably based on the processes of the

We copy or modify these processes to execute them in vitro.

A

Central Dogma of Molecular Biology

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2
Q

Central Dogma of Molecular Biology processes

A

replication, transcription and translation

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3
Q

Major Stages in DNA Replication

A

Formation of the Replication Fork
Primer binding
Elongation (DNA polymerase)
Termination

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4
Q

a technique that amplifies a specific DNA segment into billions of copies, enabling scientists to study a small DNA sample in detail.

does not have the same enzymes as the naturally occuring dna replication

A

POLYMERASE CHAIN REACTION (PCR)

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5
Q

POLYMERASE CHAIN REACTION (PCR) parts

A

dna template
primers
Taq polymerase
dNDPs
Buffer/Cofactors

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6
Q

POLYMERASE CHAIN REACTION (PCR) parts

Short DNA sequences that serve as the starting point of the amplification.
Determines w/c portion of the genome is to be sequenced.
Specificity of the PCR process depends on its design

A

primers

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7
Q

POLYMERASE CHAIN REACTION (PCR) parts
is a heat-stable version of the DNA pol enzyme used

A

Taq polymerase

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8
Q

enzymes that need Mg2+ as cofactors

A

Polymerases

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9
Q

END-POINT ANALYSIS OF PCR types

A

gel electrophoresis, NA hybridization (blotting) and Sequencing, Northern Blot (RNA), Southern Blot (DNA)
Sanger Sequencing

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10
Q

POLYMERASE CHAIN REACTION (PCR) process

A

Sample of the project
DNA extraction
Thermal cycler
Gel Electrophoresis (or other END-POINT ANALYSIS OF PCR)
DNA sequencing

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11
Q

Thermal cycler temperature for
denaturation and DNA separation fork

A

94

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12
Q

Thermal cycler temperature for initial denaturation

A

94

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13
Q

Thermal cycler temperature for annealing (attaches primers)

A

50-65

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14
Q

Thermal cycler temperature for extension. 1 min per kilobasepair

A

72

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15
Q

Thermal cycler temperature for elongation polymerize

A

72

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16
Q

END-POINT ANALYSIS OF PCR

a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. the molecules to be separated are pushed by an electrical field through a gel that contains small pores.

A

Gel Electrophoresis

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17
Q

Gel Electrophoresis process

A

PCR DNA amplicons on agarose gel
Eletric current
DNA separation
UV transillumination and documentation
DNA amplicaons show up in bands
DNA ladder will be used as reference
POSITIVE results: DNA bands of the same size and position as the DNA ladder/ marker DNA

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18
Q

END-POINT ANALYSIS OF PCR

a laboratory technique that detects RNA in a biological sample, such as blood or tissue, to study gene expression

purified RNA fragments from a biological sample (such as blood or tissue) are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments.

A

Northern Blot (RNA)

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19
Q

Northern Blot (RNA) process

A

RNA extraction
electrophoresis
RNA separated by size
Northern blotting
labeled probes (binds gene of interest, complement)
Visualization of labeled RNA on X-ray film or fluroscent labeled

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20
Q

END-POINT ANALYSIS OF PCR

a method for hybridizing one or more labeled DNA probes to a large number of DNA fragments and discriminating among them. The procedure depends on the ability of denatured DNA single strands to bind tightly to nitrocellulose under certain conditions.

A

Southern Blot (DNA)

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21
Q

Southern Blot (DNA) process

A

Nitrocellulose filter on PCR samples
Blotting paper to dry
the DNA transferred to filter
then Hybridize with unique nucleic acid prob to be a complementary DNA sequence
Expose XRAY to filter
autoradiogram

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22
Q

END-POINT ANALYSIS OF PCR

is a method for determining the sequence of DNA nucleotides in a specific region of the genome. It’s also known as the “chain-termination method”.

A

Sanger Sequencing

23
Q

Sanger Sequencing process

A

denaturing dsDANA with heat
making multiple copies of a segment
add primers
add to 4 polymerase solutions
add complimentary chain on segments until termination
denature the grown chains and eletrophorese

24
Q

Sanger Sequencing 4 polymerase solutions

A
  1. ddATPS, dATPs, dCTPs, dGTPs, dTTPs
  2. ddTTPs, dATPs, dCTPs, dGTPs, dTTPs
  3. ddCTPs, dATPs, dCTPs, dGTPs, dTTPs
  4. ddGTPs, dATPs, dCTPs, dGTPs, dTTPs
25
Q

DIFFERENT TYPES OF PCR

A

Reverse Transcription PCR (RT-PCR)

Quantitative PCR (qPCR) or real time PCR (rt-PCR)

RT-qPCR

26
Q

DIFFERENT TYPES OF PCR
most of the time this is coupled with qPCR thus called RT-qPCR.

A

Reverse Transcription PCR (RT-PCR)

27
Q

This has been very common during the pandemic since COVID-19 has ssRNA genome.

A

Reverse Transcription PCR (RT-PCR)

28
Q

Why do we need to create cDNA template? Why not just
use RNA?

A

RNA is fragile and easily degraded, making it difficult to study gene expression.

29
Q

Reverse Transcription PCR (RT-PCR) process

A

reverse transcription: RNA of Sample DNA transcribed to complimentary DNA or cDNA

amplification: Amplify cDNA by PCR (replication of copies)

30
Q

DIFFERENT TYPES OF PCR

Simultaneous amplification plus quantification of the DNA target.
Results can be viewed real-time.

A

Quantitative PCR (qPCR) or real time PCR (rt-PCR)

31
Q

The higher the initial concentration of the DNA target in Quantitative PCR (qPCR) or real time PCR (rt-PCR)

A

The higher the initial concentration of the DNA target, the earlier it will be detected
(based on cycle number)

32
Q

indicates how many times a machine needed to try to copy a particular genetic material before being able to detect in Quantitative PCR (qPCR) or real time PCR (rt-PCR)

A

CT (cycle threshold)

33
Q

can be looked at as an indirect indicator of the amount of viral genetic material detected from a particular specimen on a particular test at a particular time in Quantitative PCR (qPCR) or real time PCR (rt-PCR)

A

CT value

34
Q

DIFFERENT TYPES OF PCR
Quantitative PCR (qPCR) or real time PCR (rt-PCR) 2 types

A

Fluorescent Dye-Based qPCR
probe-based qPCR

35
Q

Quantitative PCR (qPCR) or real time PCR (rt-PCR) 2 types

the fluorophore intercalates to the new DNA which emits light (signal) when it gtes excited

A

Fluorescent Dye-Based qPCR

36
Q

Quantitative PCR (qPCR) or real time PCR (rt-PCR) 2 types

DNA probe binds with the target sequence and is typically quenched (inactivated), but when DNA elongates, the fluorophore separates from quencher molecule. The fluorophore can now emit a light signal

A

probe-based qPCR

37
Q

PCR Have been widely used in COVID 19 testing

A

RT-qPCR

38
Q

DIFFERENT TYPES OF PCR

Combination of reverse
transcription and quantitative PCR

Very useful in estimating gene
expression levels

A

RT-qPCR

39
Q

RT-qPCR process

A

sample
RNA isolation: Isolate the RNA
Reverse transcription: Use reverse transcriptase to convert the RNA into cDNA
cDNA amplification: Amplify the cDNA using a thermal cycler
Detection: Use a fluorescent chemistry to detect the cDNA in real-time
Data analysis: Analyze the data

40
Q

APPLICATIONS OF PCR

Some genes are only found in a particular species/group of bacteria.

Some genes are universal in all organisms but has some level of variability w/c can be used for characterization

A

Detection/characterization of microorganisms

41
Q

genes that are universal in all organisms but has some level of variability Example

A

● 16s rRNA gene or 16s rDNA in prokaryotes
● internal transcribed spacer (ITS) in fungi

42
Q

genes that are only found in a particular species/group of bacteria. Example

A

● IS6110, MBP64 genes in M. tuberculosis
● Nuc gene encodes for thermonuclease in S. aureus
● B-glucuronidase gene in E. coli

43
Q

is a technique that measures the mass of molecules in a sample:

revolutionized the method of microorganism identification

A

MALDI-TOF

44
Q

MALDI-TOF steps

A

Sample proteins, applied to target, is overlaid with matrix solution and allowed to dry

Spot on target is pulsed with a laser

Desorbed ionized molecules accelerated by a potential difference fly through a high-vacuum and field free flight tube as a vapor

Time of flight, based on mass of particles, is captured on detector

Resulting spectrum is compared to library containing patterns of clinically relevant species

45
Q

MALDI-TOF parts

bacterial culture, mostly proteins, nucleic acids etc

A

Analyte

46
Q

MALDI-TOF parts

This is where the analyte/sample (bacterial culture) is applied or mixed with. Analyte particles will become ionized.

A

Matrix

47
Q

MALDI-TOF parts

Pulses the matrix resulting in the vaporization of the analyte and matrix mixture

A

Laser

48
Q

MALDI-TOF parts

Ionized molecules of the analyte travel inside a vacuum tube. The analyte particles will travel depending on their masses.

A

Flight-tube

49
Q

in MALDI-TOF, Time-of-flight generates what

A

mass spectra

50
Q

MALDI-TOF
The spectra is generated based on the ______________ ratio of the particles present in the analyte

A

mass/charge ratio

51
Q

MALDI-TOF
Spectra patterns are _________ for each microorganism.

that is why the spectrum is matched to a database (library) w/c contains spectra of various identified bacteria.

A

unique

52
Q

MALDI-TOF
captures mostly __________ proteins

A

ribosomal

53
Q

MALDI-TOF
% of a bacterial dry cell weight is ribosomal proteins

A

60-70%

LBYBI13 (16 decks)

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