Difference between revisions of "Molecular pathology"
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====Karyotyping==== | ====Karyotyping==== | ||
What? | |||
*Metaphase nuclei. | *Metaphase nuclei. | ||
How? | |||
*Chromosomes are identified by: | *Chromosomes are identified by: | ||
**Size (chromosome 1 = largest, chromosome 22 = smallest). | **Size (chromosome 1 = largest, chromosome 22 = smallest). | ||
**Position of centromere. | **Position of centromere. | ||
**Banding pattern - | **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> | ***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: C-banding (C=constitutive heterchromatin), G-banding (G=Giemsa | ****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: | Image: | ||
*[http://commons.wikimedia.org/wiki/File:NHGRI_human_male_karyotype.png Karyotype (WC)]. | *[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==== | ====ISH==== | ||
Revision as of 14:01, 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.
- The (PCR) primers are designed bind to the mutated sequence.
- Technique for finding a (specific) single base change.
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.
Cytogenetics
General
- Large changes (chromosomal).
- Maximum resolution 3-4 megabase pairs (3-4 million base pairs); may be less - dependent on band density.[4]
- Morphologic data.
Techniques
- ISH = in situ hybridization.
- FISH = fluorescent in situ hybridization.
- SISH = silver in situ hybridization.[5]
- 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:[6]
- 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.
- Examples: Q-banding (Q=Quinacrine), C-banding (C=constitutive heterchromatin), G-banding (G=Giemsa), R-banding (R=reverse).
- Several different techniques (stains) are used:[6]
Image:
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:
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,[7] yet the announcement is in pay-for-access journal (Nature Structual Biology).[8]
Wnt/beta-catenin pathway
Important in hepatoblastomas.[9]
See also
References
- ↑ 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.
- ↑ 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.
- ↑ 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.
- ↑ Humphrey, Peter A; Dehner, Louis P; Pfeifer, John D (2008). The Washington Manual of Surgical Pathology (1st ed.). Lippincott Williams & Wilkins. pp. 695. ISBN 978-0781765275.
- ↑ URL: http://www.immunoportal.com/modules.php?name=News&file=article&sid=186. Accessed on: 2 May 2011.
- ↑ URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC355922/. Accessed on: 10 May 2011.
- ↑ Worldwide Protein Data Bank. URL: http://www.wwpdb.org/faq.html Accessed on: April 22, 2009.
- ↑ 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.
- ↑ 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.