Difference between revisions of "Molecular pathology"
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! Name of technique | ! Name of technique | ||
! Key elements | ! Key elements | ||
! Type of change | ! Type of change detected | ||
! Cost | ! Cost | ||
! Other | ! Other | ||
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| gel electrophoresis, hybridization probe with label | | gel electrophoresis, hybridization probe with label | ||
| useful for finding a specific known change, quantifying gene copy number | | useful for finding a specific known change, quantifying gene copy number | ||
| $$$ | |||
| | |||
|- | |||
| Amplification-refractory mutation system (ARMS) | |||
| PCR with mutation-specific primer, gel electrophoresis | |||
| useful for finding a specific known change | |||
| $$ | |||
| primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product | |||
|} | |||
A comparison of ISH and karyotyping: | |||
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto" | |||
! Name of technique | |||
! Key elements | |||
! Type of change detected | |||
! Cost | |||
! Other | |||
|- | |||
| ISH break apart probe | |||
| 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 | |||
| $$$ | |||
| can detect translocations - without knowing the specific fusion product | |||
|- | |||
| ISH fusion probe | |||
| probes label different genes (that are not adjacent) | |||
| translocation involving the two genes labeled; one may find: gene duplication, loss of a gene) | |||
| $$$ | |||
| can detect one specific translocation | |||
|- | |||
| ISH one colour | |||
| probe label one region (gene) | |||
| gene duplication, loss of a gene | |||
| $$$ | |||
| | |||
|- | |||
| Karyotyping | |||
| metaphase nuclei | |||
| large scale changes (fusions, deletions, translocations) | |||
| $$$ | | $$$ | ||
| | | | ||
Revision as of 16:57, 10 May 2011
Molecular pathology is the future of pathology.
Overview
Molecular pathology can be divided as follows:
| Molecular pathology | |||||||||||||||||||
| PCR-based techniques | Cytogenetics | ||||||||||||||||||
Tabular comparisons
A comparison of common molecular techniques:
| Name of technique | Key elements | Type of change detected | Cost | Other |
|---|---|---|---|---|
| DNA sequencing | PCR, sequencing machine | any change in genome; does not account for post-transcriptional changes (one cannot definitively infer protein level change) | $$$$ | gold standard |
| RNA sequencing | reverse transcription PCR, sequencing maching | any change in the mRNA (post-splicing); useful for infering protein level changes | $$$ | less costly than DNA sequencing (no extrons) |
| Restriction fragment length polymorphism (RFLP) | PCR, restriction endonuclease digestion, gel electrophoresis | useful for finding common base pair changes | $$ | value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene |
| Southern blot | gel electrophoresis, hybridization probe with label | useful for finding a specific known change, quantifying gene copy number | $$$ | |
| Amplification-refractory mutation system (ARMS) | PCR with mutation-specific primer, gel electrophoresis | useful for finding a specific known change | $$ | primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product |
A comparison of ISH and karyotyping:
| Name of technique | Key elements | Type of change detected | Cost | Other |
|---|---|---|---|---|
| ISH break apart probe | probes label two parts of a (normal) gene; the two markers straddle (common) break points | gene fragmentation consistent with translocation; one may find: loss of part of the gene, gene duplication | $$$ | can detect translocations - without knowing the specific fusion product |
| ISH fusion probe | probes label different genes (that are not adjacent) | translocation involving the two genes labeled; one may find: gene duplication, loss of a gene) | $$$ | can detect one specific translocation |
| ISH one colour | probe label one region (gene) | gene duplication, loss of a gene | $$$ | |
| Karyotyping | metaphase nuclei | large scale changes (fusions, deletions, translocations) | $$$ |
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.