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Difference between revisions of "Molecular pathology"
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{{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}} | {{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}} | ||
{{familytree | |,|-|-|^|-|-|.|}} | {{familytree | |,|-|-|^|-|-|.|}} | ||
{{familytree | B11 | | | | B12 |B11= | {{familytree | B11 | | | | B12 |B11=PCR-based<br>techniques|B12=[[Cytogenetics]]}} | ||
{{familytree/end}} | {{familytree/end}} | ||
</center> | </center> | ||
== | ==PCR-based techniques== | ||
===General=== | ===General=== | ||
What? | |||
*Very small changes - submicroscopic. | *Very small changes - submicroscopic. | ||
* | **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. | ||
*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. | ||
== | ==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> | ||