Difference between revisions of "Molecular pathology"

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(split-out cytogenetics)
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==Cytogenetics==
==Cytogenetics==
===General===
{{Main|Cytogenetics}}
*Large changes (chromosomal).
This deals with karyotyping and ISH.
**Maximum resolution 3-4 megabase pairs (3-4 million base pairs); may be less - dependent on band density.<ref>{{Ref WMSP|695}}</ref>
*Morphologic data.
 
===Techniques===
*ISH = in situ hybridization.
**FISH = fluorescent in situ hybridization.
**SISH = silver in situ hybridization.<ref>URL: [http://www.immunoportal.com/modules.php?name=News&file=article&sid=186 http://www.immunoportal.com/modules.php?name=News&file=article&sid=186]. Accessed on: 2 May 2011.</ref>
*Karyotyping.
 
====Karyotyping====
What?
*Metaphase nuclei.
 
How?
*Chromosomes are identified by:
**Size (chromosome 1 = largest, chromosome 22 = smallest).
**Position of centromere.
**Banding pattern - using (special) stains:
***Several different techniques (stains) are used:<ref>URL: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC355922/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC355922/]. Accessed on: 10 May 2011.</ref>
****Examples: Q-banding (Q=Quinacrine), C-banding (C=constitutive heterchromatin), G-banding (G=Giemsa), R-banding (R=reverse).
*****Q-banding is the standard at UHN.
 
Image:
*[http://commons.wikimedia.org/wiki/File:NHGRI_human_male_karyotype.png Karyotype (WC)].
 
Notes:
*Quinacrine dye - AT-rich regions brighter than GC-rich regions.
 
====ISH====
*Usu. interphase nuclei.
 
May be prepared from:
*In situ paraffin sections (section 4 micrometers thick).
*Isolated nuclei (section 10 micrometers thick).
*Cytospin.
*Cultured cells.
 
=====Probe types=====
Types:
*Fusion (two colours).
*Breakapart (two colours).
*Deletion/duplication (e.g. trisomy).
 
Image:
*[http://commons.wikimedia.org/wiki/File:Bcrablmet.jpg Bcr-Abl translocation (WC)].


==Other garbage==
==Other garbage==

Revision as of 14:26, 10 May 2011

Molecular pathology is the future of pathology.

Overview

Molecular pathology can be divided as follows:

 
 
 
Molecular
pathology
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Molecular
techniques
 
 
 
Cytogenetics

Molecular

General

  • Very small changes - submicroscopic.
  • Sequence data.

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.
  • Southern blot.
    • Analysis of proteins.
  • 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.

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.[2]
    • DNA is extracted using phenol and isopropanol.[3]

Cytogenetics

Main article: Cytogenetics

This deals with karyotyping and ISH.

Other garbage

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,[4] yet the announcement is in pay-for-access journal (Nature Structual Biology).[5]

Wnt/beta-catenin pathway

Important in hepatoblastomas.[6]

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. ↑ 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.
  3. ↑ 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.
  4. ↑ Worldwide Protein Data Bank. URL: http://www.wwpdb.org/faq.html Accessed on: April 22, 2009.
  5. ↑ 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.
  6. ↑ 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

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