Overview of Genomic Technologies in Clinical Diagnostics Flashcards
1
Q
List some genomic technologies used today
A
- PCR
- Fragment analysis
- Sanger Sequencing
- Fluorescence in situ hybridisation (FISH)
- Array - comparative genomic hybridization (Array CGH)
- Multiplex ligation-dependent probe amplification (MLPA)
- Next-Generation sequencing
2
Q
How does PCR work
A
- Fundamental for many DNA applications 3 step process
- PCR is used to amplify a specific region of
DNA - Primers flank the region you want to amplify
- Each cycle doubles the amount of DNA
copies of your target sequence - Amplify enough DNA molecules so that we have sufficient material for downstream
applications
3
Q
How can we use PCR for fragment analysis?
A
- PCR followed by
capillary electrophoresis - Here we are sizing the
PCR product - Can be used to detect
repeat expansions or
other small size changes
(up to a few hundred bp)
4
Q
Explain how we can diagnose Repeat expansion diseases
A
- Huntington’s disease – severe
neurodegenerative disorder - Caused by CAG repeat expansion in the
Huntingtin (HTT) gene - Normal < 27 copies; Intermediate 27-35
copies; Pathogenic > 35 copies - Expanded protein is toxic and accumulates
in neurons causing cell death - Diagnosed with fragment analysis
5
Q
How does Sanger sequencing work
A
- Cycle Sequencing; based on the same principles
as PCR - Each of the 4 DNA nucleotides has a different
dye so we can determine the nucleotide
sequence. - Up to 800bp of sequence per reaction; Good for sequencing single exons of the gene
- Slow, low-throughput and costly to perform for
large numbers of samples
6
Q
How can we use Sanger sequencing to detect mutations
A
- We can identify single nucleotide
polymorphisms (SNPs), or
mutations - Detection of a mutation in a family by use of Sanger
Sequencing - R1042G mutation in gene C3
segregates with affected
individuals - Mutation causes disease cutaneous vasculitis
7
Q
What is Fluorescent in situ hybridisation
A
- To detect large chromosomal
abnormalities - Extra chromosomes
- Large deleted segments
- Translocations
8
Q
How does FISH work
A
1) Design Fluorescent probe to
chromosomal region of interest
2) Denature probe and target DNA
3)Mix probe and target DNA
(hybridisation)
4) Probe binds to target
5) Target fluoresces or lights up
9
Q
How does Array CGH work
A
- Similar to microarray technology
- Array comparative genomic
hybridisation - For detection of sub-microscopic
chromosomal abnormalities - Patient DNA labelled Green
- Control DNA labelled Red
10
Q
How do we analyse Array CGH
A
- Patient array comparative genomic
hybridisation profile - Increased green signal over a chromosomal segment in the patient DNA
- Indicates a gain in the patient sample not present in the parent
11
Q
What is MPLA
A
- Multiplex ligation-dependent
probe amplification (MLPA) is a variation of PCR that permits the amplification of multiple targets - Each probe consists of two
oligonucleotides which
recognize adjacent target sites on the DNA - We use MLPA to detect
abnormal copy numbers at specific chromosomal locations - MLPA can detect sub-microscopic (small) gene deletions/partial
gene deletions
12
Q
How can we use MLPA to analyse for mutations?
A
- Perform fragment analysis (capillary electrophoresis) of MLPA product
- An important use of MLPA is to determine
relative ploidy (how many chromosome copies?)
as specific locations - For example, probes may be designed to target
various regions of the chromosome of a human cell - The signal strengths of the probes are
compared with those obtained from a reference
DNA sample known to have two copies of the
chromosome
13
Q
How does MPLA work
A
- One probe oligonucleotide contains the sequence recognized by the forward primer, and the other contains the sequence recognized by the reverse primer.
- Only when both probe oligonucleotides are hybridized to their respective targets, can they be ligated into a complete prob
14
Q
What is Next Generation Sequencing
A
Wider range of tests in a shorter time for less money
Current strategy: Disease panels:
- Enriching to sequence of only the known disease genes
relevant to the phenotype - Panels expandable to include new genes as they are
published - Potentially pathogenic variants confirmed by Sanger
sequencing
15
Q
What is Exome sequencing
A
- There are ~21,000 genes in the human genome
- Often we are only interested in the gene protein-coding exons or ‘exome’ represents 1-2% of the genome
- Some ~80% pathogenic mutations are protein coding
- More efficient to only sequence the bits we are interested in, rather than the entire genome
- Costs £1,000 for a genome, but only £200-£300 for an exome
