Molecular pathology
Revision as of 13:30, 24 May 2011 by Michael (talk | contribs) (→Tabular comparisons: +another table)
Molecular pathology is the future of pathology.
Overview
Molecular pathology can be divided as follows:
| Molecular pathology | |||||||||||||||||||
| PCR-based techniques | Cytogenetics | ||||||||||||||||||
Tabular comparisons
Overview
A simplified overview of molecular pathology:
| Name of technique | Advantages | Disadvantages |
|---|---|---|
| in situ hybridization (ISH) | good way to find gene losses and duplications (one colour) and gene splits and fusions (two colours); intermediate resolution - better resolution than karyotyping for specific target | target specific (if the target is wrong no information is gained or one is mislead by negative result); NOT good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong |
| karyotyping | finds large scale gains, losses and rearrangements; good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong | low resolution (completely misses small scale changes) |
| PCR + sequencing or enzyme digestion and electrophoresis | high resolution (can find very small changes, e.g. base pair substitutions) | expensive; thus, limited to small regions; sequencing is the gold standard but very costly; enzyme digestion and electrophoresis is a compromise of sorts where one needs to know something about the expected abnormality |
PCR-based techniques
A comparison of common molecular techniques:
| Name of technique | Key elements | Type of change detected | Cost | Other |
|---|---|---|---|---|
| DNA sequencing | PCR, sequencing machine | any (small) DNA change in the genome; does not account for post-transcriptional changes (one cannot definitively infer protein level change) | $$$$ | gold standard; will not detect large scale changes unless the break points/fusion regions are sequenced |
| RNA sequencing | reverse transcription PCR, sequencing maching | any change in the mRNA (post-splicing); useful for infering protein level changes | $$$ | less costly than DNA sequencing - as extrons are not sequenced |
| Restriction fragment length polymorphism (RFLP) | PCR, restriction endonuclease digestion, gel electrophoresis | useful for finding common base pair changes | $$ | value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene |
| Southern blot | gel electrophoresis, hybridization probe with label | useful for finding a specific known change, quantifying gene copy number | $$$ | does not use PCR - included here as it doesn't fit elsewhere |
| Amplification-refractory mutation system (ARMS) | PCR with mutation-specific primer, gel electrophoresis | useful for finding a specific known change | $$ | primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product |
Cytogenetics
A comparison of ISH and karyotyping:
| Name of technique | Key elements | Type of change detected | Cost | Other |
|---|---|---|---|---|
| ISH break apart probe (two colours) | probes label two parts of a (normal) gene; the two markers straddle (common) break points | gene fragmentation consistent with translocation; one may find: loss of part of the gene, gene duplication | $$$ | can detect translocations - without knowing the specific fusion product |
| ISH fusion probe (two colours) | probes label different genes (that are not adjacent) | translocation involving the two genes labeled; one may find: gene duplication, loss of a gene) | $$$ | can detect one specific translocation |
| ISH probe (one colour) | probe label one region (gene) | gene duplication, loss of a gene | $$ | |
| Karyotyping | metaphase nuclei | large scale changes (fusions, deletions, translocations) | $$$ | gives the "big picture" view of all the (nuclear) DNA |
PCR-based techniques
General
What?
- Very small changes - submicroscopic.
- Changes in sequence
Techniques
- DNA sequencing.
- Real time-PCR, AKA real time-quantitative PCR (RQ-PCR).
- RNA sequencing.
- May be examined after reverse transcription (RNA -> DNA), i.e. RT-PCR.
- Amplification-refractory mutation system (ARMS):[1]
- Technique for finding a (specific) single base change.
- The (PCR) primers are designed bind to the mutated sequence.
- If the mutation is present a PCR product is seen.
- If the mutation is absent no PCR product is seen.
- The (PCR) primers are designed bind to the mutated sequence.
- Technique for finding a (specific) single base change.
- Restriction fragment length polymorphism (RFLP).[2]
- Technique useful for finding a single base change.
- Restriction endonuclease(s), generally, will generate different fragment lengths if nucleotide change is present.
- This techique is most useful if one is looking for a specific (small) genetic change (e.g. F5 Arg534Gln).
- Technique useful for finding a single base change.
Specific tests
A list of tests are found in the Molecular pathology tests article.
DNA & RNA extraction
- Techniques are largely standardized.
- Protocols exist for fresh tissue and formulin fixed parafin imbeded tissue.
Other molecular tests
Techniques
- Southern blot.
- DNA quantification.
Key elements:
- Gel electrophoresis.
- Labeling with hybridization probe.
Cytogenetics
Main article: Cytogenetics
This deals with karyotyping and ISH.
Miscellaneous stuff
World protein databank
I can't help think it is ironic that the protein databank goal is to maintain a free and publicly available archive,[5] yet the announcement is in pay-for-access journal (Nature Structual Biology).[6]
Wnt/beta-catenin pathway
Important in hepatoblastomas.[7]
See also
References
- ↑ Little S (May 2001). "Amplification-refractory mutation system (ARMS) analysis of point mutations". Curr Protoc Hum Genet Chapter 9: Unit 9.8. doi:10.1002/0471142905.hg0908s07. PMID 18428319.
- ↑ URL: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml. Accessed on: 10 May 2011.
- ↑ Chomczynski P, Sacchi N (2006). "The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on". Nat Protoc 1 (2): 581–5. doi:10.1038/nprot.2006.83. PMID 17406285.
- ↑ Pikor LA, Enfield KS, Cameron H, Lam WL (2011). "DNA extraction from paraffin embedded material for genetic and epigenetic analyses". J Vis Exp (49). doi:10.3791/2763. PMID 21490570.
- ↑ Worldwide Protein Data Bank. URL: http://www.wwpdb.org/faq.html Accessed on: April 22, 2009.
- ↑ Berman H, Henrick K, Nakamura H (December 2003). "Announcing the worldwide Protein Data Bank". Nat. Struct. Biol. 10 (12): 980. doi:10.1038/nsb1203-980. PMID 14634627.
- ↑ Cotran, Ramzi S.; Kumar, Vinay; Fausto, Nelson; Nelso Fausto; Robbins, Stanley L.; Abbas, Abul K. (2005). Robbins and Cotran pathologic basis of disease (7th ed.). St. Louis, Mo: Elsevier Saunders. pp. 923. ISBN 0-7216-0187-1.