IMMUNOLOGIC AND MOLECULAR DIAGNOSTICS II Flashcards
Molecular-based diagnostic methods are notably based on the processes of the
We copy or modify these processes to execute them in vitro.
Central Dogma of Molecular Biology
Central Dogma of Molecular Biology processes
replication, transcription and translation
Major Stages in DNA Replication
Formation of the Replication Fork
Primer binding
Elongation (DNA polymerase)
Termination
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
POLYMERASE CHAIN REACTION (PCR)
POLYMERASE CHAIN REACTION (PCR) parts
dna template
primers
Taq polymerase
dNDPs
Buffer/Cofactors
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
primers
POLYMERASE CHAIN REACTION (PCR) parts
is a heat-stable version of the DNA pol enzyme used
Taq polymerase
enzymes that need Mg2+ as cofactors
Polymerases
END-POINT ANALYSIS OF PCR types
gel electrophoresis, NA hybridization (blotting) and Sequencing, Northern Blot (RNA), Southern Blot (DNA)
Sanger Sequencing
POLYMERASE CHAIN REACTION (PCR) process
Sample of the project
DNA extraction
Thermal cycler
Gel Electrophoresis (or other END-POINT ANALYSIS OF PCR)
DNA sequencing
Thermal cycler temperature for
denaturation and DNA separation fork
94
Thermal cycler temperature for initial denaturation
94
Thermal cycler temperature for annealing (attaches primers)
50-65
Thermal cycler temperature for extension. 1 min per kilobasepair
72
Thermal cycler temperature for elongation polymerize
72
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.
Gel Electrophoresis
Gel Electrophoresis process
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
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.
Northern Blot (RNA)
Northern Blot (RNA) process
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
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.
Southern Blot (DNA)
Southern Blot (DNA) process
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
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”.
Sanger Sequencing
Sanger Sequencing process
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
Sanger Sequencing 4 polymerase solutions
- ddATPS, dATPs, dCTPs, dGTPs, dTTPs
- ddTTPs, dATPs, dCTPs, dGTPs, dTTPs
- ddCTPs, dATPs, dCTPs, dGTPs, dTTPs
- ddGTPs, dATPs, dCTPs, dGTPs, dTTPs
DIFFERENT TYPES OF PCR
Reverse Transcription PCR (RT-PCR)
Quantitative PCR (qPCR) or real time PCR (rt-PCR)
RT-qPCR
DIFFERENT TYPES OF PCR
most of the time this is coupled with qPCR thus called RT-qPCR.
Reverse Transcription PCR (RT-PCR)
This has been very common during the pandemic since COVID-19 has ssRNA genome.
Reverse Transcription PCR (RT-PCR)
Why do we need to create cDNA template? Why not just
use RNA?
RNA is fragile and easily degraded, making it difficult to study gene expression.
Reverse Transcription PCR (RT-PCR) process
reverse transcription: RNA of Sample DNA transcribed to complimentary DNA or cDNA
amplification: Amplify cDNA by PCR (replication of copies)
DIFFERENT TYPES OF PCR
Simultaneous amplification plus quantification of the DNA target.
Results can be viewed real-time.
Quantitative PCR (qPCR) or real time PCR (rt-PCR)
The higher the initial concentration of the DNA target in Quantitative PCR (qPCR) or real time PCR (rt-PCR)
The higher the initial concentration of the DNA target, the earlier it will be detected
(based on cycle number)
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)
CT (cycle threshold)
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)
CT value
DIFFERENT TYPES OF PCR
Quantitative PCR (qPCR) or real time PCR (rt-PCR) 2 types
Fluorescent Dye-Based qPCR
probe-based qPCR
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
Fluorescent Dye-Based qPCR
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
probe-based qPCR
PCR Have been widely used in COVID 19 testing
RT-qPCR
DIFFERENT TYPES OF PCR
Combination of reverse
transcription and quantitative PCR
Very useful in estimating gene
expression levels
RT-qPCR
RT-qPCR process
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
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
Detection/characterization of microorganisms
genes that are universal in all organisms but has some level of variability Example
● 16s rRNA gene or 16s rDNA in prokaryotes
● internal transcribed spacer (ITS) in fungi
genes that are only found in a particular species/group of bacteria. Example
● IS6110, MBP64 genes in M. tuberculosis
● Nuc gene encodes for thermonuclease in S. aureus
● B-glucuronidase gene in E. coli
is a technique that measures the mass of molecules in a sample:
revolutionized the method of microorganism identification
MALDI-TOF
MALDI-TOF steps
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
MALDI-TOF parts
bacterial culture, mostly proteins, nucleic acids etc
Analyte
MALDI-TOF parts
This is where the analyte/sample (bacterial culture) is applied or mixed with. Analyte particles will become ionized.
Matrix
MALDI-TOF parts
Pulses the matrix resulting in the vaporization of the analyte and matrix mixture
Laser
MALDI-TOF parts
Ionized molecules of the analyte travel inside a vacuum tube. The analyte particles will travel depending on their masses.
Flight-tube
in MALDI-TOF, Time-of-flight generates what
mass spectra
MALDI-TOF
The spectra is generated based on the ______________ ratio of the particles present in the analyte
mass/charge ratio
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.
unique
MALDI-TOF
captures mostly __________ proteins
ribosomal
MALDI-TOF
% of a bacterial dry cell weight is ribosomal proteins
60-70%
