Difference between revisions of "Molecular pathology"

From Libre Pathology
Jump to navigation Jump to search
(more)
(+a table)
Line 10: Line 10:
{{familytree/end}}
{{familytree/end}}
</center>
</center>
===Tabular comparisons===
A comparison of common molecular techniques:
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
! Name of technique
! Key elements
! Type of change
! 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
| $$
| value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene
|-
| Southern blot
| gel electrophoresis, hybridization probe with label
| useful for finding a specific known change, quantifying gene copy number
| $$$
|
|}


==PCR-based techniques==
==PCR-based techniques==

Revision as of 16:26, 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 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 $$ value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene
Southern blot gel electrophoresis, hybridization probe with label useful for finding a specific known change, quantifying gene copy number $$$

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

  • Southern blot.
    • Analysis of proteins.

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

  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

Retrieved from "https://librepathology.org/w/index.php?title=Molecular_pathology&oldid=5285"