Difference between revisions of "Molecular pathology"

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(split-out cytogenetics)
(more)
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{{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}}
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{{familytree | B11 | | | | B12 |B11=Molecular<br>techniques|B12=[[Cytogenetics]]}}
{{familytree | B11 | | | | B12 |B11=PCR-based<br>techniques|B12=[[Cytogenetics]]}}
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==Molecular==
==PCR-based techniques==
===General===
===General===
What?
*Very small changes - submicroscopic.
*Very small changes - submicroscopic.
*Sequence data.
**Changes in sequence


===Techniques===
===Techniques===
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*RNA sequencing.
*RNA sequencing.
**May be examined after reverse transcription (RNA -> DNA), i.e. RT-PCR.
**May be examined after reverse transcription (RNA -> DNA), i.e. RT-PCR.
*Southern blot.
**Analysis of proteins.
*Amplification-refractory mutation system (ARMS):<ref name=pmid18428319>{{cite journal |author=Little S |title=Amplification-refractory mutation system (ARMS) analysis of point mutations |journal=Curr Protoc Hum Genet |volume=Chapter 9 |issue= |pages=Unit 9.8 |year=2001 |month=May |pmid=18428319 |doi=10.1002/0471142905.hg0908s07 |url=}}</ref>
*Amplification-refractory mutation system (ARMS):<ref name=pmid18428319>{{cite journal |author=Little S |title=Amplification-refractory mutation system (ARMS) analysis of point mutations |journal=Curr Protoc Hum Genet |volume=Chapter 9 |issue= |pages=Unit 9.8 |year=2001 |month=May |pmid=18428319 |doi=10.1002/0471142905.hg0908s07 |url=}}</ref>
**Technique for finding a (specific) single base change.
**Technique for finding a (specific) single base change.
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****If the mutation is present a PCR product is seen.   
****If the mutation is present a PCR product is seen.   
****If the mutation is absent no PCR product is seen.
****If the mutation is absent no PCR product is seen.
 
*Restriction fragment length polymorphism (RFLP).<ref>URL: [http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml]. Accessed on: 10 May 2011.</ref>
**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====
====Specific tests====
A list of tests are found in the ''[[Molecular pathology tests]]'' article.
A list of tests are found in the ''[[Molecular pathology tests]]'' article.
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**RNA is usually extracted with acid guanidium thiocyanate, phenol and choroform.<ref>{{cite journal |author=Chomczynski P, Sacchi N |title=The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on |journal=Nat Protoc |volume=1 |issue=2 |pages=581–5 |year=2006 |pmid=17406285 |doi=10.1038/nprot.2006.83 |url=}}</ref>
**RNA is usually extracted with acid guanidium thiocyanate, phenol and choroform.<ref>{{cite journal |author=Chomczynski P, Sacchi N |title=The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on |journal=Nat Protoc |volume=1 |issue=2 |pages=581–5 |year=2006 |pmid=17406285 |doi=10.1038/nprot.2006.83 |url=}}</ref>
**DNA is extracted using phenol and isopropanol.<ref>{{cite journal |author=Pikor LA, Enfield KS, Cameron H, Lam WL |title=DNA extraction from paraffin embedded material for genetic and epigenetic analyses |journal=J Vis Exp |volume= |issue=49 |pages= |year=2011 |pmid=21490570 |doi=10.3791/2763 |url=}}</ref>
**DNA is extracted using phenol and isopropanol.<ref>{{cite journal |author=Pikor LA, Enfield KS, Cameron H, Lam WL |title=DNA extraction from paraffin embedded material for genetic and epigenetic analyses |journal=J Vis Exp |volume= |issue=49 |pages= |year=2011 |pmid=21490570 |doi=10.3791/2763 |url=}}</ref>
==Other molecular tests==
*Southern blot.
**Analysis of proteins.


==Cytogenetics==
==Cytogenetics==
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This deals with karyotyping and ISH.
This deals with karyotyping and ISH.


==Other garbage==
==Miscellaneous stuff==
===World protein databank===
===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,<ref>Worldwide Protein Data Bank. URL: [http://www.wwpdb.org/faq.html http://www.wwpdb.org/faq.html] Accessed on: April 22, 2009.</ref> yet the announcement is in pay-for-access journal (''Nature Structual Biology'').<ref name=pmid14634627>{{cite journal |author=Berman H, Henrick K, Nakamura H |title=Announcing the worldwide Protein Data Bank |journal=Nat. Struct. Biol. |volume=10 |issue=12 |pages=980 |year=2003 |month=December |pmid=14634627 |doi=10.1038/nsb1203-980 |url=}}</ref>
I can't help think it is ironic that the protein databank goal is to maintain a free and publicly available archive,<ref>Worldwide Protein Data Bank. URL: [http://www.wwpdb.org/faq.html http://www.wwpdb.org/faq.html] Accessed on: April 22, 2009.</ref> yet the announcement is in pay-for-access journal (''Nature Structual Biology'').<ref name=pmid14634627>{{cite journal |author=Berman H, Henrick K, Nakamura H |title=Announcing the worldwide Protein Data Bank |journal=Nat. Struct. Biol. |volume=10 |issue=12 |pages=980 |year=2003 |month=December |pmid=14634627 |doi=10.1038/nsb1203-980 |url=}}</ref>

Revision as of 15:59, 10 May 2011

Molecular pathology is the future of pathology.

Overview

Molecular pathology can be divided as follows:

 
 
 
Molecular
pathology
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PCR-based
techniques
 
 
 
Cytogenetics

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

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