7- Future Of Genomic Medicine

Question 1

How is CF tested for?

Answer 1

Neonatal heelprick test
•Biochemical test for immunoreactive trypsinogen
•PCR with primers that span region of CFTR gene for 3bp deletion that results in ΔF508

Question 2

What is Sanger sequencing?

Answer 2

DNA sequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication. DNA fragments separated by electrophoresis to determine sequence. Took a very long time.
Can now use capillary electrophoresis , producing a chromatography, to speed up process.

Question 3

Give next generation sequencing examples

Answer 3

Massively parallel sequencing, bridge amplification, emulsion PCR

Question 4

What is Nanopore technology

Answer 4

Dna is isolated and fed through a Nanopore. Depending on nucleotide, different electrical impulse generated.

Question 5

What are implications of genetic testing in mono genetic and complex disease.

Answer 5

Monogenetic
Carrierstatusinformatione.g.Tay Sachs,cysticfibrosis
o Detectionofrareresinousconditionsinnewbornse.g.MCADdeficiency o Geneticcounselling(mustbeprovided)
o Determinationoflateronsetdiseasee.g.hereditarybreastcancer
For complex disease
o Maycausealarm
o Mayofferfalsereassurance
o Dataprivacyconcerns
o Limitedclinicalutilityastreatmentsarerare

Question 6

What are limitations of NGS

Answer 6

Cost is still high
Library biases
Data overload
Hard to determine carrier vs driver mutations
Read depth (mutated genes less expressed in genes)
More personalised medicine causes less returns for pharmaceutical companies
Need good quality clinical data for genotype-phenotype correlation

Question 7

HOw does CRISPR/CAS9 work?

Answer 7

Developed from bacterial mechanism to cleave viral genome to prevent bacteriophage infection
•Cas9 a prokaryotic exonuclease that is directed by a guide RNA
Can target almost any sequence in any genome as the PAM sequence (5’-NGG-3‘) is so ubiquitous

Question 8

What are limitations of CRISPR

Answer 8

Off target effects
•PAM sequences are ubiquitious in genome
•Cas9-guide RNA complexes bind to imperfectly matched sequence
•May need to target gRNA delivery to specific cells
•Viability of cells after injection
•Kang et al., 2016
•4/26 embryos modified but some mosaicism