Difference between revisions of "Molecular pathology"

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===Tabular comparisons===
===Tabular comparisons===
====Overview====
A simplified overview of molecular pathology:
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
! 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====
====PCR-based techniques====
A comparison of common molecular techniques:
A comparison of common molecular techniques:

Revision as of 13:30, 24 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

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.
  • 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