[M] Lec 05: Molecular Diagnostics (Molecular Methods) Flashcards
Enumerate the molecular methods
- PCR
- Gel electrophoresis
- qPCR
- RT PCR
Enumerate the DNA sequencing methods
Sanger
Next Gen
DNA Microarray
Pyrose
PCR key components
- Integral component which contains the
gene to be copied - An enzyme responsible for sequentially
adding free nucleotides conferring or - Act as a template for the new DNA ; used in copying specific region in DNA
- DNA template
- DNA polymerase
- DNA primers
PCR is repeated approximately how many times?
30
Steps of PCR
- Denaturation
- Annealing
- Extension
PCR phases
- Double-stranded DNA (dsDNA) strands are separated into 2 single strands by the use of heat.
- DNA polymerase enzyme synthesizes new complementary strands by adding individual deoxynucleotides to the 3’ end of the primer to continue copying the template molecule
- Oligonucleotide primers are recombined with the original ssDNA.
- Denaturation
- Extension
- Annealing
PCR phases
- 72°C (162°F), can range from 68–78°C.
- 95°C (203°F), can range from 90–98°C
- 55°C (131°F), can range from 30–70°C.
- Extension
- Denaturation
- Annealing
Refers to:
- Detection of gene mutations in early stages of cancer.
○ Identification of viral DNA associated with specific cancers such as:
▪ Human Papillomavirus (HPV)
▪ Herpes Simplex Virus (HSV) type 1 and 2
▪ Varicella Zoster Virus (VZV)
▪ Cytomegalovirus (CMV)
▪ Epstein-Barr Virus (EBV)
▪ Japanese Encephalitis Virus - It can also detect bacterial infection such as Chlamydia pneumonia and Mycoplasma spp.
PCR
Refers to:
- Allows macromolecules
such as the DNA, RNA fragment, or protein in a
mixture to be separated according to their molecular size and/or charge. - Molecules to be separated are placed in sample “wells” in a thin porous gel slab, covered by a buffered solution and placed in a horizontal electrophoresis chamber
Gel electrophoresis
○ Cations containing a positive charge are attracted to ________ which has a negative charge.
○ Anode containing a negative charge are attracted to ______ which has a positive charge.
- Cathode
- Anode
○ _______ containing a positive charge are attracted to cathode which has a negative charge.
○ ________ containing a negative charge are attracted to anode which has a positive charge.
- Cations
- Anions
Factors affecting the rate of gel electro migration
Size of DNA molecule
Voltage applied
Presence of ethidium bromide (fluorescent dye)
Electrophoresis buffer
Agarose gel concentration
Gel electro buffers
○ Tris, borate and EDTA
○ TAE Tris-acetate-EDTA
○ SDS
Agarose gel conc
- better for separating larger DNA fragments.|
- good for splitting smaller DNA fragments
- Lower percent
- Higher percent
Agarose types
- General-purpose agarose
- It is genetic quality tested grade
- Ideal for DNA recovery
Molbio agarose
Agarose types
- Easy to handle
- Flexible even at high gel percentages
- Excellent sieving and highest gel
strength of all the agaroses
PCR agarose
Agarose types
- Used for restriction enzyme digests,
ligation and transformation - High sieving capacity
PCR low-melt agarose
Agarose types
- Used for embedding chromosome
- Pulsed field electrophoresis of mega
base DNA - Contains bromophenol blue for
monitoring electrophoresis.
Low-melt agarose
Agarose types
Separation of large DNA fragments
Pulsed filed gel agarose (PFGE)
Agarose types
- Used for Immunoelectrophoresis (IEP)
and Isoelectric focusing (IEF) - To determine relative molecular
weight (MW) such as antibodies, serum
electrophoresis.
Agarose for IEP and IEF
Parts of gel electrophoresis
- Molecules with similar size travel to similar
location - When the DNA is separated by size in gel
electrophoresis, they will appear as a band in
the gel.
Band
Parts of gel electrophoresis
This is a fluorescent dye to see the DNA
Ethidium bromide
Parts of gel electrophoresis
- Each band represents a pre-determined length of DNA (measured in base pairs or bp)
- This contains multiple bands in one lane.
DNA ladder
Parts of gel electrophoresis
- Loading of sample in wells
- This also consists of loading well. This is
where you load the DNA sample. - The DNA will migrate in a single vertical
lane towards the positive charge. Since the
DNA has a negative charge, it will migrate
to the positive charge.
Lane
Refers to:
- Most widely used methods for quantitative estimation of gene expression.
- PRINCIPLE: Amplification of DNA in real-time and measured by a fluorescent probe.
- It is used to detect the presence of pathogens and determine the number of copied DNA sequences of interest.
Real time PCR or quantitative PCR (qPCR)
Parts of qPCR
SYBR green dye
Hydrolysis probes
qPCR
- used as an intercalating dye that binds to
dsDNA product and emits a fluorescent signal - Involves Fluorescent Resonance Energy Transfer (FRET); the emitted fluorescent dye is reduced by the presence of another dye called quencher in close proximity.
- SYBR Green dye
- Hydrolysis probes
Refers to:
- Combination of a reverse transcription PCR with quantitative PCR
- Used for rapid detection of gene expression changes.
RT-qPCR
RT-qPCR, define
- An enzyme responsible for the
formation of DNA from RNA. - Short synthetic DNA transcribed from a
specific mRNA using reverse transcriptase - A single-strand converted to double
strand by reverse transcriptase PCR
(RT-PCR) - Composed of short sequences of
thymine nucleotides - A string of adenine nucleotides
- Used for separating DNA from
homogenized tissue samples by
washing with a solvent
- Reverse transcriptase
- Complimentary DNA
- RNA
- Oligo primers
- Poly (A) tail
- Elution
Key components of RT-qPCR
- Using elution column
- Synthesis of CDNA
Refers to:
- It is considered as the GOLD STANDARD FOR GENETIC
DIAGNOSIS. - It is developed by FREDERICK SANGER in 1977.
- It is the fundamental for identification of mutation as it can
determine a relatively small amount of human DNA fragment
Sanger sequencing
Key components of Sanger sequencing
DNA Polymerase enzyme
Primers
ssDNA template
Dideoxynucleotide triphosphates
Sanger sequencing
- Short pieces ssDNA that binds to DNA template and acts as a “starter” for the polymerase.
- Produced by DNA amplification which also utilizes modified fluorescently labeled nucleotide bases known as dideoxynucleotide triphosphates.
- Final nucleotide fragment is labeled with a
_____________.
- Primers
- ssDNA template
- Fluorochrome
Study steps of Sanger
Please lang
Refers to:
- Sequence only protein-coding regions of the genome called EXOME (exome sequencing
Next generation sequencing
In NGS, this aids in identifying uknown diseases causing mutation, wherein the data goes through stringent analysis
Exome sequencing
Steps in NGS
- Template preparation
- Library amplification
- Sequencing
- Data analysis
In NGS, what are the two procedures involved in library amplification
Emulsion PCR
Bridge PCR
Library amplification (Emulsion/Bridge)
- PCR denatures library fragment leading 2 separate strands: one Reverse Strand that anneals to the beads.
- DNA is then attached tot he surface of the cell while the other DNA end strand attach to primers creating bridges structures
- Emulsion PCR
- Bridge PCR
NGS (Sequencing)
Define
- Amplifying cluster’s of ssDNA fragments
- Chemically modified nucleotides bind to DNA template strand wherein each nucleotide contains a fluorescent tag
- Blocks the fusion or merging of the next phase or next nucleotide.
- Cluster generation
- Sequencing by synthesis
- Reversible terminator
NGS (Data analysis)
Instrument software is used to identify nucleotides (a process called _________).
This anlogrithm translates the color of each cluster at a specific sequencing by synthesis
Base ceiling
Refers to:
● A technology that accelerates genetic analysis.
● Uses a DNA chip, which is similar to microprocessors that
speed up computation product of bonding or direct synthesis
of specific DNA probes on a stationary silicon-based support.
DNA Microarray
These are miniature gene fragments attached to glass chips
Microarrays
Application of the sequencing technique include:
- Comparative genomic hybridization
- Detection of extremely low proteins (CHONS) concentration
DNA Microarray
Refers to:
PRINCIPLE: Detects the release of pyrophosphate when nucleotides are added to the DNA chain.
Pyrose sequencing
Pyrose sequencing
- Normal dAT is replaced by _______
- This enzyme uses ATP to produce light
- Substrates needed
- Enzymes needed
- dATPas (deoxyadenosine alpha theotriphosphate)
- Luciferase
- Adenosine Phosphosulphate, Luciferin
- ATP sulfurylase, Luciferase, Apyrase
Refers to:
- DNA in here is sliced or split at specific locations
through the use of enzymes - Uses restriction endonucleases
`Strand cleavage methods
Refers to:
The application of this method is for: investigating small genomes such as microorganisms or plasmids and if there is a change in the nucleotide sequence or mutation.
Strand cleavage methods
Refers to:
- Considered as a 3rd generation PCR
- This uses a water-oil emulsion droplet technology that was developed in 201
Droplet digital PCR
Size of signle droplet in Droplet Digital PCR
20, 000 nanoliters
Refers to:
- Has the ability to detect and quantify virus in samples in very low viral copy numbers.
- Reagents are similar for qPCR
- Template amplification occurs in each droplet and analyzed for fluorescence
Droplet digital PCR
Digital PCR amplification
Give end color for:
1. Positive result
2. Negative result
- Green
- Gray
Methods of Amplification
- Strand displacement amplification
- Nucleic Acid Amplification
- Hybridization
Refers to:
- A fully automated method which amplifies target nucleic acid without the use of a thermocycler.
- It uses uniform isothermal temperature (37°C - 55°C)
- Amplified products are detected by turbidity or color change.
- Uses a series of primers, DNA polymerase and restriction enzymes.
Strand displacement amplifcation
Refers to:
○ Diagnose STIs such as Chlamydia and Gonorrhea
○ Performed by using a swab (endocervical/ urethral)from a patient or noninvasively on a urine sample.
Strand displacement amplification
Refers to:
- Detect low levels or amount of DNA orRNA
- Amplification is based on targeted regions of viral RNA or DNA
- Used for screening blood bags and resolve false reactive
donations on serological methods
NAAT
Refers to:
- It is the process by which two complementary single-stranded DNA or RNA molecules bond together to form a double-stranded molecule.
Hybridization
Two types of hybridization probes
Fluorescent and chromogen
It is a small purified ssDNA or RNA with a known sequence and used to identify the presence of a complementary DNA or RNA sequence in an unknown sample
Probes
Probes are based on what principle?
It is based on the PRINCIPLE OF COMPLEMENTARITY. A probe can be tagged with a fluorescent dye (green and red) and can also be a mix of 2 different colors, which gives rise to a yellow color called a fusion signal.
Probe composition
- It can be made from one sequence.
- It is a mixture of two or more sequences.
- It can be DNA or RNA.
- Homogenous
- Heterogenous
- Type of nucleic acid
Probes can be labeled by what?
- Radioactive Isotopes
- Nonradio-Isotopic Molecules
The ff are examples of which type of probe labels
○These are haptens.
○Digoxigenin, Alkaline Phosphatase, Biotin, or a
Fluorescent Compound
○Probes labeled with biotin-detected by specific Abs.
Non-radioisotopic molecules
Methods to detect nonradiolabeled probes
Direct and indirect
Refers to
- It detects and locates specific DNA sequences on a chromosome.
- It utilizes nucleic acid probes to identify DNA probes varying in size (few thousand to hundreds of thousands) of long bases covalently attached to a fluorescent.
Fluorescence in situ hybridization
Refers to:
- This is also sensitive for identifying specific chromosomal translocation and numerical chromosome alteration such as T cell lymphomas, B cell malignancies and Graft-versus-host disease
- Large chromosomal regions can be analyzed, decreasing false negative that is pivotal to the nature of the PCR.
FISH
Blotting methods
- Southern blot analysis
- Northern blot analysis
- Western blot analysis
Blotting method
- PRINCIPLE: To measure the size and amount of a specific
DNA sequences in a mixture. - Detection of a specific DNA fragment.
Southern blot
Pertains to a membrane wherein the biological molecules are absorbed or immobilized.
Blot
A process of moving the molecules from a gel to membrane followed by a detection on the membrane.
Blotting
Southern blot considerations of method
Pls study
Blotting methods
- Target is DNA
- Target is RNA
- Target are proteins
- Southern
- Northern
- Western
Refers to
- The target molecules are proteins.
- The principle is to detect antibodies to specific epitopes of antigen subspecies.
- KEY ELEMENTS: Horseradish Peroxidase Probe–an enzyme probe.
Western blot analysis
Methods of detection western blot
- Probing: Direct detection using a Primary Antibody
- Indirect detection using a Secondary Antibody
