Basic Viro Flashcards
PCR principles
1985 Kerry Mullis
Primer mediated enzymatic amplification of DNA.
Using a DNA polymerase to synthesise a strand of DNA complementary to the template strand.
Components of PCR
- DNA template
- DNA polymerase - Taq polymerase that doesn’t denature 98 C and can function at optimum temp of 70 C
- Oligonucleotide primers (20 to 30 bp) complementary to 3’ ends of the sense and anti-sense strands of the target sequence.
- Deoxynucleotide triphosphates: A,T,G, and C
- Buffer: magnesium and potassium for stability
PCR steps
- Denaturation: heating mixture to 94 C for 15-30 seconds. DNA denatured into single strands.
- Annealing: temp lowered to 54-60C for 20-40 seconds. Primers bind (anneal) to the simple mentally sequences of template DNA
- Elongation/Extension: 72-80C. Polymerase adds bases to each 3’ primer and extends the DNA sequence in 5’ to 3’ direction.
- DNA binding Dyes: most commonly used is SYBR green which non specifically binds to dsDNA and exhibits fluorescence 1000-fold when binding.
Advantages: simple design, can be used for multiple genes, lower cost
Disadvantages: lack of specificity - binds to all ds DNA. Cannot be used in multiplex (parallel reaction mixtures need setting up).
- PCR primer-probe based detection: use some form of fluorescence quenching to ensure target specific fluorescence is detected only when the amplicon of interest is present.
Primer/target-specific oligonucelotide prove us labelled with a reporter fluorophore and it’s fluorescence is quenched when the specific target is NOT present. This is by covalentlu attaching a quencher molecule to the primer or prove and there exists a mechanism by which the reporter and quencher separate when primer/probe binds to the target.
Eg: Taqman probes - hydrolysis assay. Has a fluorescent reporter at 5’ and a quencher at 3’ end. Common reporter-quencher pairs at fluorescein (FAM) enoting green fluorescence and black hole quencher 1 dye.
Serotypes
A serotype is defined as a variation within a microbial species, distinguished by the humoral immune response. The serotype classification of bacteria or viruses is based on their surface antigens and was established before the availability of other techniques, such as genome sequencing or mass spectrometry. Antibodies generated to one serotype do not usually efficiently protect against another serotype. Serotypes have been described in many viral species and generally correspond to genotypes.
Transcription vs translation
The purpose of transcription is to make RNA copies of genes. The purpose of translation is to synthesize proteins for cellular functions. Translation produces proteins, while transcription produces mRNA, tRNA, rRNA, and non-coding RNA. In prokaryotes, translation and transcription occurs in the cytoplasm
LAMP
loop-mediated isothermal amplification of DNA or RT-LAMP for RNA.
The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets.
the target sequence is amplified at a constant temperature of 60–65 °C
Uses Bst – Bacillus stearothermophilus – DNA polymerase.
Generations of PCR
The first generation of PCR relies on gel electrophoresis to analyze PCR products, but always challenged by low detection limit, laborious operation, and single application (qualitative). The second generation of PCR, also called real-time quantitative PCR (RT-qPCR), can quantify the products with standard curves, but also show low tolerance to interfering substances [1]. Digital PCR (dPCR) is the third generation of PCR that enables absolute quantification through partitioning the reaction. Highly sensitive and accurate in molecular detection, this technology has demonstrated applications like trace DNA detection, rare mutation detection and copy number variation
Digital PCR
Digital PCR builds on traditional PCR amplification and fluorescent-probe–based detection methods to provide highly sensitive absolute quantification of nucleic acids without the need for standard curves.
In the Droplet Digital™ PCR System, a PCR sample is partitioned into 20,000 droplets. After amplification, droplets containing target sequence are detected by fluorescence and scored as positive, and droplets without fluorescence are scored as negative. Poisson statistical analysis of the numbers of positive and negative droplets yields absolute quantitation of the target sequence.
Advantages:
1. Small sample quantity
2. Sensitive and specific
3. Less sensitive to inhibition
4. copy number variation (CNV) analysis
Intrathecal Ab testing
Ab index = viral ab ratio/ albumin ratio
Viral ab ratio = CSF ab/serum ab in paired sample
Albumin ratio = CSF albumin/ serum albumin
Ratio < 3 is normal
Raised controls (eg rubella, VZV, HSV) indicates a polyclonal response within the CNS
Antibody avidity testing
2 wells
1st well: bound antigen to plate + test serum - washed with 0.85% NACL, then anti-human IgG HRP conjugate added. In acute & chronic infection, antigen antibody complex will NOT be disrupted.
2nd well: bound antigen to plate + test serum - washed with 8M urea, then anti-human IgG HRP conjugate added. In acute infection, Ab-Ab complex will be disrupted and ab will be washed away. In chronic, this will not happen due to stronger bond.
Avidity index = OD Urea/OD NACL x 100%. Eg for CMV, 80% is the cut off used to say high vs low avidity.
What is an open reading frame?
In molecular biology, reading frames are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible reading frames will be “open” (the “reading”, however, refers to the RNA produced by transcription of the DNA and its subsequent interaction with the ribosome in translation). Such an ORF may contain a start codon (usually AUG in terms of RNA) and by definition cannot extend beyond a stop codon (usually UAA, UAG or UGA in RNA). That start codon (not necessarily the first) indicates where translation may start. The transcription termination site is located after the ORF, beyond the translation stop codon. If transcription were to cease before the stop codon, an incomplete protein would be made during translation
Sanger sequencing
AKA chain termination method
3 main steps:
- DNA sequence for chain termination PCR:
*The DNA sequence of interest is used as a template for a special PCR called chain termination PCR (CTP).
* CTP works like a standard PCR except for the addition of modified nucleotides (dNTPs) called dideoxyribonucleotides (ddNTPs).
*In standard PCR, during extension phase, DNA polymerase adds dNTPs to the growing chain by catalysing formation of phosphodiester bond between the free 3’OH of the last nucleotide with the 5’phosphate of the next nucleotide. SEE FIGURE IN PHONE ALBUM.
* In CTP, ddNTPs lack the 3’OH group required for binding - so when ddNTPs are incorporated by the DNA polymerase at random, chain termination occurs.
* Millions to billions of oligonucleotode copies of the DNA sequence of interest are made at random lengths terminated by ddNTPs. - Size separation by gel electrophoresis:
*The oligonucleotides are separated by gel electrophoresis.
*DNA samples are loaded into one end of a gel matrix and electric current applied.
*As DNA is negative charged, it’ll be pulled towards the positive electrode on the opposite side.
* The speed of movement will depend on the charge which is determined by the size.
* Smaller fragments move quicker.
- Gel analysis and determination of DNA sequence
*Reading the gel to determine the sequence of the input DNA.
* As DNA polymerase only makes DNA in 5’ to 3’ direction from a starting primer, each terminal ddNTP will correspond to a specific nucleotide in the original sequence.
*Reading gel bands from smallest to largest will determine the 5’ to 3’ sequence of the original DNA.
Reading a Sanger sequence:
Depends on which of the two complementary DNA strands is of interest and what primer is available. For eg: if the strands are A & B and the strand of interest is A, but the primer is more suited to strand A, the output will be a sequence of strand B and then the computer has to convert the letters to the sequence of strand A.
Basic DNA structure
- Double helix
- Each strand is made of string of molecules called deoxyribonucleotides (dNTPs).
- Each dNTP contains a phosphate group, sugar group, and 1 of 4 nitrogenous bases (A,T,G,C).
- dNTPs are strung together in a linear fashion by phosphodiester bonds between sugar of one dNTP (3’OH) and the phosphate group of the next.
- The nucleotides are bound together by hydrogen bonds between complementary bases.
SEE FIGURE IN ALBUM
Types of NGS
- WGS
- Targeted metagenomics
- Shotgun metagenomics
Generations of sequencing
1st gen - Sanger
2nd gen - Pyrosequencing - measuring pyrophosphate synthesis instead of fluorescently labelled nucleotides
3rd gen - ability to perform single molecule sequencing which means there is NO NEED for DNA amplification step. Higher throughput and longer reads being sequenced. Eg: Oxford nanopore
