Overview of Genomic Technologies in Genetic Diagnosis Flashcards
Genomic Technologies in Genetic Diagnosis include…
- PCR
- Fragment Analysis
- Sanger Sequencing
- Fluorescence in situ hybridisation (FISH)
- Array CGH
- Multiplex ligation-dependant probe amplification (MLPA)
- Next-Generation Sequencing
What is PCR?
A technique used to amplify a specific region of DNA
How does PCR work?
Primers flank the region you want to amplify and DNA Polymerase use that primer to extend nucleotides.
Each cycle doubles the amount of DNA copies of your target sequence
What is fragment analysis?
Fragment analysis is a technique to separate the DNA fragments produced in PCR by capillary electrophoresis
-sizing the PCR product
Uses of fragment analysis
Used to detect:
- repeat expansions
- other small size changes (up to a few hundred base pairs)
Example of a repeat expansion disease
Huntington’s disease
What is Huntington’s disease and what is it caused by?
A severe neurodegenerative disorder
-caused by CAG repeat expansion in the Huntington (HTT) gene, resulting in an expanded protein toxic to neurones causing cell death
Normal (<27 copies)
Intermediate (26-35 copies)
Pathogenic (>35 copies)
The more repeats you have, the more severe the disease
How is Huntington’s disease diagnosed?
Fragment analysis
What is Sanger Sequencing?
Target DNA is copied many times, making fragments of different lengths (same principles as PCR)
Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence from that.
Features of sanger sequencing
Sequences up to 800bp of sequence per reaction (good for sequencing single exons of genes)
Slow, low through-put and costly to perform for large numbers of samples
What can Sanger Sequencing be used for?
Identification of single nucleotide polymorphisms (SNPs) or mutations
Fluorescence in situ hybridization (FISH)
A fluorescence-labelled probe detects specific nucleotide sequences within cultured cells on a microscopic slide (in metaphase spread).
1) Design fluorescent probe to the chromosomal region of interest
2) Denature probe and target DNA
3) Mix probe and target DNA (hybridisation)
4) Target fluoresces and lights up
5) Can observe it under a microscope
Uses of FISH
Used to detect large chromosomal abnormalities:
- extra chromosomes (e.g. trisomy 21)
- large deleted segments
- translocations
Array Comparative Genomic Hybridization (CGH)
Microarray technique used to identify copy number variations (CNVs)- duplications & deletions
Patient DNA labelled green
Control DNA labelled red
If there is equal hybridisation between patient and control, the green and red cancel each other out and we get no signal.
Green signal= more patient DNA
Red signal= loss of patient DNA
Multiplex ligation-dependent probe amplification (MLPA)
Variation of PCR which permits amplification of multiple DNA targets using a single probe which consists of two oligonucleotides
oligonucleotides recognise adjacent target sites on the DNA
Use of MLPA
To detect abnormal copy numbers at specific chromosomal locations
-relative ploidy (how many chromosome copies at specific locations)
To detect sub-microscopic gene deletions/partial gene deletions
How does MLPA work?
Denaturation of dsDNA
One probe oligonucleotide contains the DNA sequence recognised by the forward primer, the other contains the DNA sequence recognised by the reverse primer.
Only when both probe nucleotides are hybridised to their respective targets, can they be ligated into a complete probe.
Then we can perform an amplification step.
Then you perform fragment analysis (capillary electrophoresis) of MLPA product, and the signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome
Next Generation Sequencing
Features of NGS
group of automated techniques used for rapid DNA sequencing
-replaced Sanger sequencing for almost all sequencing tests in the lab
Wider range of tests in a shorter time for less money
Uses of Next Generation Sequencing
Disease Panel Test
- enriching to sequence only the known disease genes relevant to the phenotype
- panels expandable to include new genes as they are published
- potentially pathogenic variants are confirmed by Sanger sequencing
Exome sequencing (NGS)
A strategy of sequencing only the coding regions of a genome
More efficient to only sequence exome because ~80% of pathogenic mutations are protein coding
Costs £1000 for a genome, but only £200-£300 for an exome
How does exome sequencing work?
Target Enrichment
1) We make NGS library and hybridise it with baits which are RNA sequences complimentary to the gene exons
2) In the hybridisation reactions, any sequences which are complementary will bind to one another
3) Baits with biotin tags on them can capture these specific DNA sequences with streptavidin coated magnetic beads
4) Using a magnet, we can pull out those specific fragments
5) We can then wash away the genomic DNA we don’t want, resulting in an enriched library of our exons
Whole Genome Sequencing (WGS)- NGS
Universally accepted that genome sequencing will become commonplace in diagnostic genetics
NOT all tests will automatically move to whole genome sequencing:
>Panels/single gene tests may still be more suitable for some diseases e.g. cystic fibrosis
>Capillary-based methods: Repeat expansions, MLPA, family mutation confirmation Sanger sequencing
>Array-CGH: large sized chromosomal aberrations
Disadvantage of NGS
Result interpretation of clinical genomes (exome and whole) currently has a substantial manual component:
- 20,000 variants per coding genes ‘exome’
- 3 million variants in a whole human genome
Infrastructure and training needed (particularly IT and clinical scientists)
Whole genome sequencing is NOT trivial.
Ethical considerations for NGS (exome and genome sequencing)
· Modified patient consent process
· Data analysis pathways- inspect relevant genes first
· Strategy for reporting ‘incidental’ findings
