microbiology diagnostics SDL Flashcards
what is PCR and how does it work
- rapid and simple method for copying and amplifying specific DNA sequences
- consists of repetitive cycles of DNA meltinf, DNA annealing and DNA synthesis
- to be able to use this method it is necessary to know the sequence of a short region of DNA on each end of the larger sequence that needs to be copied and amplified
- this information is used to design two synthetic DNA oligonucleotides, each complementary to the sequence on one strand of the DNA double helix at opposite ends of the region to be amplified
- these oligonucleotides serve as primers for in vitro DNA synthesis
list the order of steps for PCR
- reaction mix is heated to around 95 degrees
- hydrogen bonds of the DNA nucleotides are broken down
- double stranded DNA will be denatured
- reaction mix is cooled down to annealing temp
- primers align with complementary sequence of the target DNA
- primers form hydrogen bonds with target DNA
- rection mix is heated to reach the optimum temp for the DNA polymerase
- heat stable DNA polymerase extends the primers in 5’->3’ directionm
- temp is raised again to around 95 degrees to start the next cycle
- process is repeated about 20-30 times each time to get doubling of the DNA between primers
outline the differences between microbial culture and PCR
microbial culture:
- detection demonstrates presence of live organisms
- sample needs to be fresh
- turnaround depends on growth rate of organism
- may require enrichment prior to detection
- generally broad
PCR:
- very sensitive detection
- evidence of presence but not certain if organisms are alive or dead
- can be broad or very specific
- sample can be frozen
- quick turn around (1-3 days)
- very sensitive to contamination
- relatively cheap
what does PCR detect
PCR directly detects pathogen DNA (or viral RNA that has been converted to DNA)
what does immunodiagnostics detect
Immunodiagnostics detect antibodies that the animal produced when comes in contact with the pathogen, which could be through exposure, infection or vaccination. In neonates, immunodiagnostics will also detect maternal antibodies taken up via colostrum. Some ELISAs and snap tests detect antigen.
an example of an immunodiagnostic test is
snap test
what is a silent mutation
substitution of one nucleotide but without a change in the amino acid due to the redundancy of the genetic code
ex. CCG -> CCA both encode for glycine
what is a missense mutation
a nucleotide substituion that leads to the change of an amino acid. new amino acid might have similar properties to the old one or the substitution is in a part of the protein that does not affect its structure or function and has no impact
what is a nonsense mutation
A point mutation that changes a codon for an amino acid into a stop codon and causes the premature termination of translation. Ifthat happens closethe 3’ end of the strand (directiosn is 5’ to 3’) than that might not have much impact on the proteins.
Most non-sense mutations lead to non-functional proteins.
Examples:A nonsense mutation is linked tohydrocephalus in Fresian horses
what is an insertion mutation
Insertion = Addition of nucleotide(s)
Deletion = Removal of nucleotide(s)
This could be a single nucleotide or several genes. The impact of that can vary enormously. If this happens in:
Non-coding region it may have no effect or changes the level of expression some genes
In promoter region it may affect the expression of a protein (higher, lower, no expression)
Original sequence ACACAGCGTGGT
New sequence ACACAGCGCGGT
what type of mutation is this and what are the consequences
This is a silent mutation the nucleotide change does not lead to a change of the amino acid the codon is for.
Original sequence ACACAGCGTGGT
New sequence ACACAGAGTGGT
what type of mutation is this and what are the consequences
This is a missense the codon goes from
CGT is the codon for arginine
AGT is the codon for serine
Original sequence ACACAGCGTGGT
New sequence ACAGCAGCGTGGT
what type of mutation is this and what are the consequences
This is a insertion of base and leads to frame shift
The new codon is arginine GCA
Then shift means you get alanine GCG then leucine TTG you can not interpret the extra T at the end.
But also tick Effect on AA Seq: major change of aa sequence downstream of mutation
Original sequence ACACAGCGTGGT
New sequence ACAACACAGCGTGGT
what type of mutation is this and what are the consequences
This is an insertion of amino acid threonine.
The rest of the amino acids are the same as it’s a 3 base insertion so no frame shift just an single amino acid insertion
Original sequence ACACAGCGTGGT
New sequence ACATAGCGTGGT
what type of mutation is this and what are the consequences
This is a frame shift BUT the new sequence is a termination it is a nonsense mutation so truncates the gene. It would stop at the codon where there is a change.
