Use Of Genomics Dagnostics Flashcards
What are the two types of genomic diagnostics?
Indirect methods such as linkage analysis
- Looking at a marker that tends to be next to (or close to) the disease allele
- Physical proximity
- Based on recombination during meiosis
- Stuff that is close together tends to stay together
A marker and a gene which is close together (physically) tends to stay together after meiotic recombination (you discussed this in high school biology)
Direct methods- everything else we discuss today
What are the two broad types of direct detection?
Methods that only detect what is being asked for (hypothesis driven)
Methods that may query (interrogate) the entire genome
What are the hypothesis driven direct detection methods?
- southern-RFLP, PCR-RFLP, PCR sizing, ASO blot
- FSISH (tag a specific place on a chromosome with a probe)
- Northern, western
- Sanger sequencing (obtains sequence as one run at a time)
What methods query/interrogate the entire genome?
- Karyotype (g-banding)
- Microarray: array CGH, SNP chip, expression arrays
- Spectral karyotype (paint each chromosome a different color)
- Next gen sequencing-(obtains sequence in simplex fashion) -note that NGS can be focused on:
1. One gene
2. A panel of related genes
3. All of the exomes (whole exome sequencing; WES)
4. All of the genome (whole genome sequencing; WGS)
How can triplet repeat expansion disorders be detected?
In this application, the size of the PCR product itself is diagnostic
- Can be measured by southern blot
- Can measure by gel electrophoresis
- Can measure by column chromatography
Important:
- Each triplet repeat disorder must have its own diagnostic test
- The repeat change is not detectable by ASO, NGS or microarray
Humans have 46 chromosomes…
Accurate counting of chromosomes was facilitated by the advent f cytogenic techniques (in the 1960s)
- G banding
- Can detect changes in chromosomal number (aneuploidy)
- Can detect very large aberrations such as deletions, duplications, insertions, translocations and other rearrangements
- Key point, the aberration must affect a band
What are the genetic test limit of G-band karyotype?
- Whole chromosome changes (Down syndrome, Turner, kleinfelter)
- Very large deletions, duplications, or rearrangements (typical value given us more than 5 Mb)
- but it depends for example:
- 10 Mb deletions have been discovered which appear normal by G-band
- 10vMb duplications have been discovered which appear normal by G-band
- but it depends for example:
- To be detected by karyotype, the banding pattern must be altered
- G-band described in GNET DLA video
What are the limits of the FISH?
Depends on size of probe
-typically FISH can detect deletions bigger than 1Mb (sometimes smaller)
- FISH probe is long piece of DNA packed with many fluorophore so that it can be seen by light microscopy
- Explained in GNET DLAA
What are the limits of Microarray CGH?
- Most can resolve 30 kBp deletion/duplication
- depends on the Array though
What is the limit if next generation sequencing?
- Base pair sequencing
- Bottle neck is with the analysis of the data
What is karyotype?
Standard karyotype analysis was developed in the 1950s (advent of G-banding)
-Became a test fir many major chromosomal abnormalities such as deletions, insertions, translocations,aneuploidy
However, G-banding is unable to resolve smaller chromosomal changes:
-Many DNA alterations are too small to be seen under light microscope using standard Geimsa stained (banding) karyotype
- The range of detection changes depending on the location. In many cases a deletion more than 5 Mb is detectable, in other cases, it must be 8,9 or even 10 Mb - it depends on the chromosomal location and and the pattern of bands
- Also, depends on skill of the person doing the preparation
- Deletions too small to be seen under the microscope by G-band karyotype are called “Microdeletion” or “submicroscopic deletions”
- Standard G-band karyotype has relatively low resolution (key point)
Why would we use CGH instead of karyotypes?
Microarray CGH has better resolution that karyotype
A 10 Mb deletion can be detected by CGH by not karyotype
Explain DNA sequencing
Sanger sequencing using P-32 labelled nucleotides
-dideoxy chain termination, radioactive, manual, slow tedious
Sequencing using fluorescent dideoxy nucleotides
-May be automated because each base is different color (this was how the human genome was sequenced starting in the late 1980s (it took a. Long time (more than 10. Years and 300 million USD)
-Each sequencing reaction is done in a separate blue
Next gen sequencing
- lasers, photos, and computers
- Massively parallel obtainment of sequence data
- Now a full human DNA sequence can be obtained in a few days
Why is data storage and analysis more challenging than DMA sequencing ?
It is now more costly to store the data from DNA sequencing projects than it is to obtain the DNA sequence data in the first place
Describe the types of next gen sequencing
The methods have been organized such that the more difficult, more expensive is mentioned at the top. This is due to the final mentions having fewer number of variants to be identified
- Whole Genome sequencing
- All. 3 billion base pairs really 6 billion because diploid)
- Because terabytes of information, and many millions of variants
- Whole exome sequencing (exome)
- Only 1% of the genome which codes for protein ; the exons (has hundreds of thousands of variants)
- Targeted gene panels
- A group of genes known to be involved in a common pathway or Pathology
- Many (if not most ) disorders have overlapping features
- Single gene analysis
- In depth analysis of a single gene
- Likely to be very few variants
