Intro to clinical genetic testing Flashcards
What do genomic laboratories perform?
Perform genetic assays
Analyse DNA or chromosomes from patients
Adult, child and prenatal referrals
Implications of results
Patients diagnosed, understand cause of problems.
Understand likely future implications (prognosis) for lifestyle choices e.g. preventative surgery for cancer
Risk to family (risk to their children) - preimplantation genetic diagnosis for future pregnancy
Personalised medicine (Pharmacogenomics) – best drug choice informed by genetics
Patietnts genetic pathway
Consultation
Variety of samples mostly blood but BM, tissue, hair root, buccal wash, urine, tissue blocks, fibroblast cultures also received.
Assessment of test very important (DNA vs RNA, direct vs full extraction), size of sample, how urgent?
Extraction of DNA salting out, RNA to cDNA to PCR
Direct mutation analysis where mutation known or common mutation, generic mutation testing where mutation not known.
Linkage where gene not yet identified or where mutations hard to find.
Results interpreted and checked
Fully interpretative report issued to referring clinician
Report
Consultation
Types of Genomic Test
SIngle gene sequencing (cyctic fibrosis - suspicion of gene)
Targeted gene panels (Next generation sequencing of set of genes linked to a condition i.e heart disease)
Exomes (Next generation sequencing of protein-coding only sequences i.e DDD study)
Genomes (Next generatino sequencing of whole genome).
Basic process of next genertion sequencing (NGS)
Start with patient DNA which is fragmented (200 nucleotides)
DNA barcoded adaptors annealed to fragments
Adaptors contain PCR primer target site - one primer set for whole reaction.
Fragments are ligated to flow cell coated with DNA sequences specific to the primer target site.
PCR reaction occurs and fragments amplified
Each DNA strand in a cluster is dervied from the one original DNA fragment.
During sequencing cycle, one fluoroscent fluorophore attached nucleotide is added to the growing strands
Laser used to excite the fluorophores and emits a signal
Optic scanner collects the signals from each fragment cluster
Builds sequence in order of fluorescent emitted
What happens once you have sequences from each fragment?
Digitally recorded
Align sequences to a reference human genome.
Basically software takes the DNA sequence and finds the best fir, in terms of sequence homology within the human genome
Whats a problem with NGS?
The masses of data it generates
How do you target only certain genes?
Library preparation
Describe library preparation (can I draw this method, do I understand?)
Use RNA probes that have been design to match the sequences of our genes of interest
Probes are biotinylated. (Biotin labelled RNA probes)
Once patient DNA has been fragmented and adaptors ligated, the RNA probes are mixed in and hybridise.
They only hybridise to the regions of interest.
The DNA and RNA mix is then passed over streptavidin coated magnetic beads.
The DNA bound to RNA sticks to the beads via the interaction of biotin and streptavidin.
Magnetism then separates the DNA bound to RNA probes from the rest of the DNA.
Following digestion to remove the RNA from DNA, we are left with a mix of DNA that is specific to the genes we are interested in.
NGS proceeds by ligating these to the flow cell.
Library preparation (basic)
Library preparation
Fragmented DNA adaptors ligated
Hybridisation
Bead separation
Wash/Elution - Result ing DNA fragments only from genes of interest
What is analysis pipeline?
a process to remove those varients we are not interested in, so we are left with a small number of candidate varients to investigate further
NGS benefits
- Allows analysis of 1 to 1000s of genes in one assay
- Whole genome sequencing (WGS) enabled
- Run multiple patients in one assay
- More cost effective
- Many more diseases diagnosed via one test
- Revolutionised NHS genomics provision
Perspectives
Genetic services evolving –
History: Cytogenetics – Genetics - Genomics
Greater integration into general medicine
Whole genome sequence for all?
Implications?
Ethics?
Cost?
Education – do healthcare professionals know enough?
Does public know enough?
How much do you want to know?
