DNA diagnostics Flashcards
Why do we use DNA for diagnostics?
- DNA is relatively stable
- DNA can be replicated in the lab (PCR) allowing for high sensitivity/ low initial sample needed
- DNA change is critical in genetic disorders, cancer
- most pathogens have DNA for genome
How do we get DNA for some DNA tests?
- no DNA purification is needed
- e.g., colony PCR of bacteria
How would you get DNA for genetic disorder tests on dogs?
- mouth swabs common (epithelial cells)
- and less commonly blood samples
When testing for non-leukocyte cancers what is required?
- biopsy
What is critical when sampling for DNA testing?
- no contamination is critical
What would you have to do for blood sample when testing for DNA?
- EDTA used as anti-coagulant as it inhibits DNases
- would not use heparin as this inhibits Taq polymerase by binding it
What has become increasingly important in disease control?
- environmental DNA sampling
What are the steps to the amplification technique for PCR?
- relies on DNA template
- specific primers to site of interest/specific to pathogen
- multiple cycles of denaturing, annealing, extension
- gel electrophoresis to reveal result
- presence/absence
Name two RNA viruses?
- influenza
- west Nile virus
For PCR with RNA viruses what is the additional step that must happen first (before the normal process)?
- Need to use reverse transcriptase enzyme
- to turn RNA > dsDNA
= reverse transcription PCR
Why do some labs prefer to use Real time or Quantitative PCR?
- for some studying we want more than just presence or absence
- also faster - no gel running - reduces time and man power
- magnitude (4x more of virus genome in this individual etc.)
- amount of genome broadly equates to amount of viable pathogen - there are exceptions though
What is a type of qPCR?
- SYBR green method is simplest
What does the SYBR green method entail?
- regular PCR reaction for one specific region
- SYBR green binds to all newly synthesised double stranded DNA
- when bound SYBR green is fluorescent
- qPCR machine measures fluorescent in tube
- detects fluorescence at the end of each denature, anneal and extend cycle
What are the uses of qPCR?
- veterinary parasitology
- molecular biology
What are the limitations to qPCR?
- contamination is an issue (all amplification methods will suffer with this)
- can only measure one (SYBR green) or < 10 (taqman) transcripts per tube
Due to the limitations of qPCR what do we use for a greater global understanding?
- SNP microarray
- next gen sequencing
What are SNP arrays a type of?
- one type of DNA microarray
What are SNP arrays heavily used in?
- heavily used in breeding programmes
What does SNP array rely on?
- relies on knowing the SNPs already from sequencing studies
What does a SNP array look like?
- glass slide with little squares containing dots that have a bit of DNA with a specific SNP - which can be used to work out what the animals alleles are
What is first added onto the SNP array?
- 100,000s of short, specific single stranded DNA oligos spotted onto glass slide
- each added at a particular grid position
What are DNA oligos (25-50bp) synthesised to represent?
- particular SNP regions
Once the DNA oligos have been added - What is the third part to a SNP array?
- Take a sample. Multiples copies of the genome (DNA from multiple cells) are sheared (split apart)
- fluorophore (dye) added to DNA fragments
- Hybridisation with SNP array - denatured to single strands to they can complement their SNP
What does a Affymetrix SNP assess?
- strong/weak hybridisation
= strong signal (fluorescence) = correct allele
= weak signal is due to not as full complementary = incorrect allele
How do illumima SNP arrays work differently to Affymetrix SNP’s?
- Still DNA hybridisation but utilising different dyes for different bases at the SNP position
What do Illumina SNP arrays determine for each sample?
- Presence/absence for each SNP
In illumina SNP array what is required to ID associations?
- computational and statistical analysis
What are the two ways we can DNA sequence?
- sanger sequencing
- next-gen sequencing
What are both sanger and next gen sequencing used for?
- sequencing specific regions
- whole genome shotgun sequencing
What are the benefits to using DNA sequencing over other methods?
- sequencing will enable understanding of relationships between samples - pathogen spread
- whole genome sequencing allows for novel mutations to be IDed
- can be especially good for mixed samples
What is sanger sequencing?
- old technique
- used for all the early sequencing projects
- horse, chicken, dog, human
What does sanger sequencing do?
- incorporation of terminator bases (ddNTPs) stops new bases being added, containing fluorophores (depending on what base) during in vitro DNA replication
What does next-gen sequencing use?
- illumina/soleca, 454, SOLiD
What are the advantages if using next-gen sequencing?
- cheaper
- far less DNA needed
What are the disadvantages for using illumina in next-gen sequencing?
- short reads
- higher error rate
What is the future of next-gen sequencing?
- future is single molecular sequencing (PacBio, Nanopore)
What are the uses of DNA sequencing?
- compare individual/breeds with whole genome sequencing
What is the aim of DNA sequencing?
- to identify base pairs within the genome associated with milk production, embryonic death, disease
What can DNA sequencing reveal?
- why cancers develop - breed specifics
- what genes specifically are causing cancers
- what drugs/ MOA would would work best
