Future of genomic medicine Flashcards
How is a DNA test performed?
1) DNA isolated from a patient sample e.g. A blood test
2) PCR used to amplify DNA quantity (+ digestion if needed) (NAGE6)
3) Visualisation on agarose gel
Why are DNA tests useful in clinic?
- Complex genetic disorders can sometimes have poly and monogenic causes, each with different treatment (GEN6)
- Translocations can produce novel proteins that act as drug targets (GEN4)
- DNA/genetics testing provides a concrete Dx of monogenic disorders such as cystic fibrosis and excludes differentials
How is gel electropheresis used in CF diagnosis?
Gel electrophoresis can be used to separate DNA of different lengths due to varied retardation by the gel, leading to the following results.
- Homozygous normal: ONE band (63bp) that moves only a short distance
- Heterozygous CF carrier: TWO bands (63 and 60bp), with the 60bp band moving further
Homozygous CF patient: ONE band (60bp), moving further than the 63bp of a normal sample
How do you test for single nucleotide changes?
1) PCR the patient DNA sample to amplify the DNA
2) Use specific restriction endonucleases on sample, one of which cuts only sequences containing the mutation
3) Use gel electrophoresis to separate the fragments based upon their size
4) Use southern blotting to transfer the fragments to a membrane
5) Add fluorescently/radioactively labelled DNA probes that will hybridise with sequences on each fragment after digestion
Analysis of the results: if the mutation is present in the DNA sample, then the endonuclease will cut at the point of the mutation, leading to a larger number of fragments and a characteristic fingerprint pattern. If heterozygous, a larger number of fragments will be visible, as shown below:
How do you test for an unknown mutation?
Sanger sequencing:
1) DNA polymerase, free nucleotides, primer and ssDNA template strand are added in equal volumes to four tubes. Each tube contains radioactively labelled dideoxynucleotide triphosphates that lack 3’ OH groups, with one tube containing labelled Adenine, another Thymine and so on
2) DNA polymerase catalyses the polymerisation of nucleotides to form a complementary DNA strand in each tube, and when a ddNTP binds, polymerisation stops and the chain is terminated (because there is no 3’ OH group do add another dNTP to)
3) This produces chains of various lengths which are then separated by gel electrophoresis using four lanes, with the shortest fragments moving furthest
4) The separated dyed fragments can be read using autoradiography to give a DNA sequence
How does next generation sequencing work?
- DNA randomly fragmented and adaptor molecules added to each end of the fragments, enabling them to bind to a fixed plate.
- DNA is first sequenced forwards, using labelled dNTPs that fluoresce a specific colour when excited by light
- As each binds, it is excited, producing a flash of light that is recorded.
- The same fragments are then turned around on the plate and sequenced backwards.
- Many different fragments are sequenced at the same time, each producing dots on a grid which are recorded.
- Forward and reverse sequences are then combined, and then the sequences of all other fragments are used to construct a sequence using overlapping regions. Any ambiguous regions are decided using a reference DNA sequence.
What is the DNA library?
the actual sample/DNA to be analysed and sequenced; this could be the whole genome or other DNA regions to answer specific questions
What are the advantages and disadvantages of Sanger and next generation sequencing
see benjis notes
How is NGS used to determine the molecular basis of monogenic diseases.
Used to rapidly sequence fragments of DNA. The library (source) selected denotes the function:
- Targeted: used to sequence a specific gene for mutation identification
- Epigenome: using bisulphite to show which regions are methylated to identify the impact on disease
- Transcriptome: converting mRNA to cDNA to sequence only transcribed proteins - e.g. If transcribed and not present in protein form, problem with translation or folding
How do advances in genomic medicine may lead to personalised/precision medicine, including the treatment of cancer.
Use of genomics to tailor medical care to individuals based on genetic makeup; this could have several uses:
- Discovery: mechanism and biomarkers of disease
- Diagnosis: identification of pathology/condition
- Classification: staging and classification of disease such as cancer
- Prognosis: certain mutations may lead to a different chance of survival
- Therapeutics: different mutations will respond to different treatment
Pharmacogenics: clinical studies using genomes of cancer patients/non-standard cancer patients can be sequenced to identify mutations for novel drug treatments
100,000 genomes project: sequencing 50k genomes of cancer patients and 50k of rare disease patients (and relatives) to identify genomic sequences
Biomarkers: molecules naturally produced that identify presence or severity of a disease or risk of conditions/events such as miscarriage
