Advanced Molecular Techniques Flashcards
What are you usually interested in when studying at DNA level
You study usually things what help with gene activity (mutations ex.)
Name 4 diagnostic/research method we can use at DNA level
- SNP –> single nucleotide
- MLPA –> Multiplexing
- CGH Array –> Looking at bigger chromosomic changes
- WES (Whole Exome Sequencing)
Which technique can be used for chromosome, gene AND exon level and which one doesn’t?
MLPA: For chromosome, Gene AND Exon changes
FISH, CGH, Karyotyping, SNP array: Usually chromosome; CGH and SNP array may be used for gene diagnostics as well
DNA sequencing only looks at Exon and q PCR looks at Exon and gene
Which technique can be used to detect copy number changes (CNV) in anything?
MLPA
What is the principle of SNP analysis?
SNP= single nucleotide polymorphisms
Oligonucleotide ligation assay. It analyses 1 mutation. The technique is bases on a KNOWN mutation where the primer doesn’t bind. While analysing the sequence, if there appears to be a gap, there is a mutation. Ligation can only work when gap is closed. So it depends on ligation
Step 1: Hybridization
Step 2: Ligation and denaturation
Step 3: PCR
What is the principle of MLPA
MLPA= Multiplex ligation dependent probe amplification, which is still depending on ligation. If there IS a mutation, there will be no gap whereas without a mutation, the sequence will have a gap.
The probe for MLPA has 3 parts:
1. Universal primer binding sequences (identical for each target)
2. Oligo/hybridization sequences which are unique so specific for target (mutation)
3. Stuffer sequence: contains of different lengths of sequence for each target (acts like a barcode but then we have to compare sizes). While seperating PCR products on gel, the fragments with the smallest stuffer sequence migrates the fastest through the agarose gel or seperating through capillary electrophoresis
Does capillary electrophoresis have a peak pattern as final result
Yes, and you can see whether the MLPA products have a homozygous - or heterozygous deletion
What can be analysed using CGH array?
CGH Array= comparitive genomic hybridization
It looks at the bigger picture of a genome, so it looks at chromosomic changes (insertions and deletions)
It has the same concept as a micro array, but instead of using cDNA, real DNA is used (also labeled with fluoresecent probes)
The CGH array compares a patient’s entire genome with that of (many) different person (Ref) who DO NOT exhibit any clinical signs with regard to the loss/gain of genetic material
Why should you use Whole EXOME sequencing instead of Whole GENOME sequencing?
You are only interested in exons which are important for gene expression/ protein hsynthesis, so it’s a targeted search. Also WGS may be too expensive and there will be a lot of unnecessary data to analyse
When should you use WES for diagnostic applications?
- When the anamnesis does NOT point into the direction of a known genetic disorder. A not yet identified gene mutation may be the case
- The patient is suspected to have a disease for which no standard diagnostic test is available or in cases the available test does not yield a conclusive answer
- If the clinical features are atypical or multi-interpretable: the differential diagnosis does not rule out multiple disorders
- When performing 1/multiple (genetic) tests is timeconsuming and/or expensive
What are the labatory steps for WES
- Isolate DNA patient
- Shear DNA to get smaller fragments
- Ligate adaptors to each fragment
- Bead capture the exon fragment (exome capture) (with oligonucleotides)
- Sequence
- Analyse
To capture genomic regions of interest, a pool of custom Oligonucleotides (probes) are synthesized and labeled with BIOTIN
What are the steps for Exome capture
- Genomic DNA is isolated and fragmented and labeled with adaptors
- To capture genomic regions of interest, a pool of custom Oligonucleotides (probes) is synthesized and labeled with beads
- The probes selectively hybridize to the genomic regions of interest (exons)
- The beads (now including the DNA fragments of interest) can be pulled down and washed to clear excess material
- The beads are removed
- The fragments can be sequenced, allowing for selective DNA sequencing of exons
- NGS –> Illumina, Ion Torrent
What are the steps for NGS (illumina)
- Template preparation –> fragmentation of DNA template
- Ligation of adaptor molecules to blunt end of template DNA
- Immunobilization of single stranded DNA template to flow cell (solid surface)
- DNA template containing adaptors are hybridized to complementary probes on surface
- Elongation of strand with dNTP
- Removal of original single stranded DNA by denaturation
- Bridge formation by hybridization to primer
- Synthesis of second strand
- Dentaturation
- Bridge formation
- Amplification
REPEAT - Bridge amplification
Million of clusters of ssDNA are generated –> population of identical templayes
- Reverse strand is removed before sequencing - Sequencing
- Cyclic reversible termination - Analysis by reading sequence and alligning
What are the steps for Cyclic reversible termination (Sequencing WES)
- Extend primer with labeled nucleotides
- Wash
- Image
- Cleave termination group and fluorescent molecules
- Wash
- Go to step 1 and repeat
What are the chromosomal anomalies
- Duplication: a gene is duplicated
- Deletion: a gene is deleted
- Inversion: different genes on chromosome are rearranged
- Translocation: part of genes on one chromosome tail gets swapped with an other gene on the other chromosome tail. The order of genes doesn’t get swapped
These alterations lead to CNV
