Lecture 5 - Molecular Diagnostics Flashcards
What are molecular diagnostics?
Various molecular biology techniques used to analyze biological markers in the genome and proteome
What are molecular diagnostics used for?
- Monitoring disease
- Detecting disease risk
- Identify therapies
Pharmacogenomics
Using molecular biology technologies to predict a response to drugs (differences in enzymes involved in metabolism)
How are molecular diagnostics used in infectious disease?
- Identify infection status
- Identify strains
How are molecular diagnostics used in cancer?
- Breast cancer risk (BRCA1/2)
- Diagnostics
- Oncogene mutations
Which factors are leading to the growth of molecular diagnostic techniques?
- Zoonotic diseases
- Animal agriculture
- Pet adoption and healthcare
- Pet insurance
Molecular diagnostic techniques for DNA/RNA
- Sanger sequencing
- SNP microarrays
- Next-generation sequencing
- PCR
- Isothermal amplification
Molecular diagnostic techniques for proteins
- ELISA (enzyme-linked immunosorbent assay)
- Proteomics
PCR is used to
- Amplify target DNA and RNA
- Test for the presence or quantity of DNA/RNA in a sample
- Targeted genotyping (single SNPs or insertion/mutation)
- Facilitate other applications such as sanger sequencing or DNA metabarcoding (by amplifying DNA)
What is needed for PCR?
- A DNA template
- A pair of synthetic single stranded DNA primers
- dNTPs (deoxyribonucleotide triphosphates)
- A heat stable polymerase (Taq)
Taq polymerase
A heat-stable polymerase from the bacteria Thermus aquaticus
Downsides of Taq polymerase
Unable to repair base pair errors during replication (cannot proofread)
The amount of DNA ____ every cycle of PCR
Doubles
How can molecular diagnostics determine disease risk?
Looks at the presence/absence of SNPs that cause disease
What is the role of the heat-stable polymerase in PCR?
To extend the primer
Binding requirements for PCR primer design
Must only bind at one location on the genome
Length requirements for PCR primer
18-25 nucleotides, helps avoid having a similar sequence elsewhere that the primer could bind to
Melting temperature requirements for PCR primer design
The temperature at which the pair of primers dissociate from the template should be within +/-5 degrees celcius
Primer dimers
When forward and backward primers bind together - must be avoided since the primer won’t work if it can bind with itself
Structure requirements for PCR primer design
Structure must be avoided
(idk what this means tbh but its in the slides so if anyone else knows let me know - Aimee)
What allows the visualization of PCR results
Gel electrophoresis
How does gel electrophoresis work?
- Separates DNA molecules by length (smaller molecules travel faster)
- DNA is loaded onto agarose gel and will result in bands at different locations which indicates where different nucleotides are located in the strand
Why is a negative control used in PCR
Contamination is an issue in PCR, a negative control ensures that the sample was not contaminated
What is capillary electrophoresis?
Capillary electrophoresis involves separating segments based on size, similar to gel electrophoresis. This can occur in the same machine as sequencing (ex. Sanger sequencing).
A limitation of PCR is that it requires prior knowledge of ____.
Some of the target sequence
List the limitations of PCR
- Requires some knowledge of the sequence
- Contamination
- Taq polymerase does not have the ability to proofread
- Some samples contain PCR inhibitors that can inhibit the reaction
- Longer segments cannot be reliably amplified
Up to what length can PCR reliably amplify?
2000bp
What are some PCR inhibitors that can be present within samples?
- Humic acid
- Urea
- Calcium ions
Which veterinary infections can PCR detect?
- Avian Influenza A
- Rift Valley Fever Porcine Viruses
- Bovine Diarrhea Virus
- Sheep & Goat Poxvirus
- African Horse Sickness
What is the abbreviated name for reverse transcription PCR
RT-PCR
What is the abbreviated name for real time PCR
qPCR aka. quantitative PCR
What is RT-PCR?
RT-PCR uses the enzyme reverse transcriptase to create a DNA template from a strand of RNA that can then be amplified.
What is RT-PCR used for?
RNA viruses or gene transcription.
Transcriptomics
A technique to look at all of the RNA in a sample. It uses the poly-A tail that is added onto RNA during transcription to amplify all of the RNA in a sample by converting it to a DNA complement.
What method is used to look for transcription of a specific gene?
RT-PCR, a specific primer is used to look for a target sequence.
What method is used to look for all RNA transcription in a sample?
RNA-seq (transcriptomics)
What is qPCR?
- Quantifies the amount of DNA in a sample.
- Uses fluorescence to determine the amount of DNA compared to a known sample.
What are the benefits of qPCR?
Faster, more sensitive, more accurate quantitation than PCR.
What is DNA metabarcoding?
It is a process that can determine which species are present in a sample with mixed DNA.
What are the steps for DNA metabarcoding?
- DNA extraction
- PCR amplification
- Next generation sequencing of PCR products
- Compare data from sequencing to a database with reference sequences
- Infer species composition
What is required for DNA metabarcoding to work?
The sequence of the species present in the sample must be in the database.
How is isothermal amplification different from normal PCR?
Isothermal amplification can occur at a constant temperature whereas PCR requires temperature cycling.
Isothermal amplification
Loop-mediated isothermal amplification is a single-tube technique for the amplification of DNA.
What technology was used in the Covid rapid test kits?
LAMP (isothermal amplification)
How does loop-mediated isothermal amplification (LAMP) work?
- 4 to 6 primers are used.
- DNA amplification occurs due to elongation of the primer which separates the target strand.
- A self-hybridizing loop forms on the new strand due to the inclusion of a reverse complimentary sequence.
- The process repeats and a loop forms on the opposite side, forming a dumbbell structure.
- The products can be detected in a variety of ways.
