Molecular pathology

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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 the specific target of the given ISH target specific (if the target is wrong no information is gained or one is mislead by the 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); requires fresh tissue/cell culture (as it is based on metaphase nuclei)
PCR + sequencing or enzyme digestion and electrophoresis high resolution (can find very small changes, e.g. base pair substitutions); can be done on formalin fixed paraffin embedded tissue 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.
  • 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).

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.
    • RNA is usually extracted with acid guanidium thiocyanate, phenol and choroform.[3]
    • DNA is extracted using phenol and isopropanol.[4]

Other molecular tests

Techniques

  • Southern blot.
    • DNA quantification.

Key elements:

  • Gel electrophoresis.
  • Labeling with hybridization probe.

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

  1. 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.
  2. URL: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml. Accessed on: 10 May 2011.
  3. 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.
  4. 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.
  5. Worldwide Protein Data Bank. URL: http://www.wwpdb.org/faq.html Accessed on: April 22, 2009.
  6. 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.
  7. 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.

External links