Difference between revisions of "Molecular pathology"

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'''Molecular pathology''' is the future of pathology.
[[Image:G-Storm thermal cycler.jpg|thumb|300px|right|A thermal cycler used for PCR-based molecular testing. ([[WC]])]]
'''Molecular pathology''' is the study of disease at the molecular level. It is becoming increasingly important in pathology. 
 
==Utility of molecular pathology==
Its utility currently includes:
# Proving clonality, esp. in hematologic malignancies, to help establish a malignant diagnosis.
# Finding recurrent genetic changes - which may be diagnostic, prognostic and suggest a specific therapy.
# Monitor minimal residual disease.  


==Overview==
==Overview==
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{{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}}
{{familytree | | | |A11| | | | |A11 =Molecular<br>pathology}}
{{familytree | |,|-|-|^|-|-|.|}}
{{familytree | |,|-|-|^|-|-|.|}}
{{familytree | B11 | | | | B12 |B11=Molecular|B12=Cytogenetics}}
{{familytree | B11 | | | | B12 |B11=Electrophoresis<br>based techniques|B12=[[Cytogenetics]]}}
{{familytree/end}}
{{familytree/end}}
</center>
</center>


==Molecular==
===Tabular comparisons===
General:
====Overview====  
*Very small changes.
A simplified overview of molecular pathology:


Techniques:
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
*DNA sequencing - RT-PCR.
! Name of technique
*RNA sequencing.
! Advantages
*Southern blot.
! Disadvantages
|-
| in situ hybridization (ISH)
| '''intermediate resolution''' - better resolution than karyotyping for the specific target of the given ISH; good way to find gene losses and duplications (one colour) and gene splits and fusions (two colours); can be done on [[formalin]] fixed paraffin embedded tissue
| '''target specific''' (if the target is wrong no information is gained ''or'' one is mislead by the negative result); NOT good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong
|-
| karyotyping
| finds '''large scale changes''' (gains, losses, rearrangements); good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong
| '''low resolution''' (completely misses small scale changes); '''requires fresh tissue/cell culture''' (as it is based on metaphase nuclei)
|-
| PCR + sequencing ''or'' enzyme digestion and electrophoresis
| '''high resolution''' (can find very small changes, e.g. base pair substitutions) - considered gold standard; can be done on [[formalin]] fixed paraffin embedded tissue
| expensive; thus, limited to small regions ('''target specific'''); enzyme digestion and electrophoresis is a compromise of sorts where one needs to know something about the expected abnormality; (gene) duplications may be difficult to prove; regions with many repeats may be difficult to sequence
|}


===Tests===
====PCR-based/electrophoresis based techniques====
Hereditary:
A comparison of common molecular techniques:
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
! Target
! Name of technique
! Technique
! Key elements
! Disease
! Type of change detected
! Notes
! Cost
! Other
|-
| DNA sequencing
| PCR, sequencing machine
| any (small) DNA change in the genome; does not account for post-transcriptional changes (one cannot definitively infer protein level change)
| $$$
| gold standard; will not detect large scale changes unless the break points/fusion regions are sequenced
|-
| RNA sequencing
| reverse transcription PCR, sequencing maching
| any change in the mRNA (post-splicing); useful for infering protein level changes
| $$$
| slightly less costly than DNA sequencing - as the extrons are not sequenced
|-
| Restriction fragment length polymorphism (RFLP)
| PCR, restriction endonuclease digestion, gel electrophoresis
| useful for finding common base pair changes
| $$
| value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene
|-
| Amplification-refractory mutation system (ARMS)
| PCR with mutation-specific primer, gel electrophoresis
| useful for finding a specific known change
| $$
| primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product
|-
|-
| F2<ref>{{OMIM|176930}}</ref>
| Southern blot
| PCR
| gel electrophoresis, hybridization probe with label
| thrombophilia
| useful for finding a specific known change, quantifying gene copy number
| see ''[[Pulmonary_embolism#Risks_factors_.28VTE.29|Risks for VTE]]''
| $$$$$
| -rarely done<br>-does '''not''' use PCR<br>-considered the gold standard for clonality<ref name=pmid10583924>{{Cite journal  | last1 = Medeiros | first1 = LJ. | last2 = Carr | first2 = J. | title = Overview of the role of molecular methods in the diagnosis of malignant lymphomas. | journal = Arch Pathol Lab Med | volume = 123 | issue = 12 | pages = 1189-207 | month = Dec | year = 1999 | doi = 10.1043/0003-9985(1999)1231189:OOTROM2.0.CO;2 | PMID = 10583924 }}</ref><br>-most labs consider fresh or frozen tissue a requirement<ref name=pmid11070157>{{Cite journal  | last1 = Reinartz | first1 = JJ. | last2 = McCormick | first2 = SR. | last3 = Ikier | first3 = DM. | last4 = Mellgen | first4 = AM. | last5 = Bonham | first5 = SC. | last6 = Strickler | first6 = JG. | last7 = Mendiola | first7 = JR. | title = Immunoglobulin heavy-chain gene rearrangement studies by Southern blot using DNA extracted from formalin-fixed, paraffin-embedded tissue. | journal = Mol Diagn | volume = 5 | issue = 3 | pages = 227-33 | month = Sep | year = 2000 | doi = 10.1054/modi.2000.19808 | PMID = 11070157 }}</ref>
|}
 
====Cytogenetics====
A comparison of ISH and karyotyping:
{| class="wikitable sortable" style="margin-left:auto;margin-right:auto"
! Name of technique
! Key elements
! Type of change detected
! Cost
! Other
|-
|-
| F5<ref>{{OMIM|612309}}</ref>
| Interphase ISH break apart probe (two colours)
| PCR
| probes label two parts of a (normal) gene; the two markers straddle (common) break points
| thrombophilia
| gene fragmentation consistent with translocation; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss)
| see ''[[Pulmonary_embolism#Risks_factors_.28VTE.29|Risks for VTE]]''
| $$$$
| can detect translocations - without knowing the specific fusion product
|-
|-
| HFE<ref>{{OMIM|613609}}</ref> Cys282Tyr, His63Asp
| Interphase ISH fusion probe (two colours)
| PCR
| probes label different genes (that are not adjacent)
| [[hemochromatosis]]
| translocation involving the two genes labeled; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss)
| -
| $$$$
| can detect one specific translocation
|-
|-
| Interphase ISH probe (one colour)
| probe labels one region (gene)
| gene duplication (or chromosomal duplication), gene loss (or chromosome loss)
| $$$
|  
|  
|
| Malignant hyperthermia
| anesthetics
|-
|-
|  
| Karyotyping
|  
| metaphase nuclei
| Herediary [[amyloidosis]]
| large scale changes (fusions, deletions, translocations)
|  
| $$$$
| gives the "big picture" view of all the (nuclear) DNA
|-
|-
|  
| Metaphase ISH probe (one colour / two colours)
|  
| probe labels one region (one colour) ''or'' probes label two parts of a (normal) gene (two colours) ''or'' probes label different genes (two colours)
| Alpha thalassemia
| gene duplication, gene loss, translocations
|  
| $$$$$
| rarely done; follows karyotyping to better characterize unusual cases; can be thought of as a karyotype and a simultaneous ISH
|}
|}


[[Lymphoma]]:
*Follicular lymphoma vs. follicular hyperplasia (BCL2).
*T cell neoplasia.
*B cell neoplasia.
*PTLPD vs. rejection.
*HHV associated lymphomas.
Leukemia:
*Several.


Carcinoma:
==Data formats==
*Nasopharyngeal (EBV quantitation).
Human gene naming is provided by the HUGO Gene Nomenclature Committee: https://www.genenames.org/
*HPV testing.
 
*Metastatic [[colorectal carinoma]].
DNA data repositories
*Non small cell [[lung cancer]].
* NCBI: National Center for Biotechnology Information
*Papillary thyroid carcinoma.
**Standard sequencing data is usually located in Nucleotide database: https://www.ncbi.nlm.nih.gov/nuccore
**Next-gen sequencing data is in short read archive: https://www.ncbi.nlm.nih.gov/sra/docs/submit/
* EMBL: European Molecular Biology Laboratory
* DDBJ: DNA Data Bank of Japan
 
DNA sequence data formats
* GenBank: human readable, can be processed by computer (fixed width, first 10 characters are an identifier).
** NCBI Reference Sequence (RefSeq) project provides sequence records and related information.
** Prefix AC_ in acession number is for genomic data, NM_ is for mRNA.
* FASTA: Sequence information
** Header starts with > and is followed by a sequence ID.
** Sequence lines should wrap always at the same width.
** Lower-case letters may indicate repetitive regions.
* FASTQ: Current standard for sequencing data
** It is essentially FASTA with quality values for the sequence.
** Quality is on a scale from 0 - 40 and represented by a distinct character.
** Upper case letters ABCDEFGHI means high quality.
** Special letters !"#$%&'()*+,-. mean low quality.
 
==Polymerase chain reaction-based techniques==
:Abbreviated ''PCR-based techniques''
:''PCR'' redirects here
===General===
*A molecular technique to duplicate DNA (or RNA) molecules ("amplify") and allow the DNA (or RNA) sequence to be determined.
 
Utility?
*Detect very small molecular changes - submicroscopic.
**Changes in sequence - may be as small as one base pair.
*Used to confirmation [[chromosomal translocation]]s that are, in clinical practice, usually found with other techniques.
 
===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):<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.
***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).<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====
A list of tests are found in the ''[[Molecular pathology tests]]'' article.


Other:
====DNA & RNA extraction====
*Oligodendroglioma.
*Techniques are largely standardized.
*Identity testing (15 STRs and amelogenin (XY) loci).
*Protocols exist for fresh tissue and [[formalin]] fixed paraffin imbedded tissue.
*Melanoma (KIT, BRAF).
**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>
*[[Synovial sarcoma]].
**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>
*Myeloproliferative disorders.
*AML, mastocytosis, GIST.


==Cytogenetics==
==Other molecular tests==
General:
===Techniques===
*Large changes (chromosomal).
*Southern blot.
**Maximum resolution 3-4 megabase pairs (3-4 million base pairs); may be less - dependent on band density.<ref>{{Ref WMSP|695}}</ref>
**DNA quantification.


Techniques:
Key elements:
*ISH = in situ hybridization.
*Gel electrophoresis.
**FISH = fluorescent in situ hybridization.  
*Labeling with hybridization probe.
**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>


Image:
==Cytogenetics==
*[http://commons.wikimedia.org/wiki/File:Bcrablmet.jpg Bcr-Abl translocation (WC)].
{{Main|Cytogenetics}}
This deals with karyotyping and ISH.


==World protein databank==
==Miscellaneous stuff==
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>
===World protein databank===
The protein databank's 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> Ironically, its announcement is in a 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>


==Wnt/beta-catenin pathway==
===Wnt/beta-catenin pathway===
Important in hepatoblastomas.<ref name=Ref_PBoD923>{{Ref PBoD|923}}</ref>
Important in hepatoblastomas.<ref name=Ref_PBoD923>{{Ref PBoD|923}}</ref>


Line 105: Line 202:
*[[Basics]].
*[[Basics]].
*[[Chromosomal translocations]].
*[[Chromosomal translocations]].
*[[DNA sequence comparison]].
*[[Tumour suppressor gene]].
*[[Next generation sequencing]].


==References==
==References==
{{reflist|2}}
{{reflist|2}}
==External links==
*[http://genome.ucsc.edu/ UCSC Genome Browser (ucsc.edu)].


[[Category:Molecular pathology]]
[[Category:Molecular pathology]]

Latest revision as of 20:24, 11 October 2023

A thermal cycler used for PCR-based molecular testing. (WC)

Molecular pathology is the study of disease at the molecular level. It is becoming increasingly important in pathology.

Utility of molecular pathology

Its utility currently includes:

  1. Proving clonality, esp. in hematologic malignancies, to help establish a malignant diagnosis.
  2. Finding recurrent genetic changes - which may be diagnostic, prognostic and suggest a specific therapy.
  3. Monitor minimal residual disease.

Overview

Molecular pathology can be divided as follows:

 
 
 
Molecular
pathology
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Electrophoresis
based techniques
 
 
 
Cytogenetics

Tabular comparisons

Overview

A simplified overview of molecular pathology:

Name of technique Advantages Disadvantages
in situ hybridization (ISH) intermediate resolution - better resolution than karyotyping for the specific target of the given ISH; good way to find gene losses and duplications (one colour) and gene splits and fusions (two colours); can be done on formalin fixed paraffin embedded tissue target specific (if the target is wrong no information is gained or one is mislead by the negative result); NOT good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong
karyotyping finds large scale changes (gains, losses, rearrangements); good for "going on a fishing expedition", i.e. looking for changes when one doesn't quite know what is wrong low resolution (completely misses small scale changes); requires fresh tissue/cell culture (as it is based on metaphase nuclei)
PCR + sequencing or enzyme digestion and electrophoresis high resolution (can find very small changes, e.g. base pair substitutions) - considered gold standard; can be done on formalin fixed paraffin embedded tissue expensive; thus, limited to small regions (target specific); enzyme digestion and electrophoresis is a compromise of sorts where one needs to know something about the expected abnormality; (gene) duplications may be difficult to prove; regions with many repeats may be difficult to sequence

PCR-based/electrophoresis based techniques

A comparison of common molecular techniques:

Name of technique Key elements Type of change detected Cost Other
DNA sequencing PCR, sequencing machine any (small) DNA change in the genome; does not account for post-transcriptional changes (one cannot definitively infer protein level change) $$$ gold standard; will not detect large scale changes unless the break points/fusion regions are sequenced
RNA sequencing reverse transcription PCR, sequencing maching any change in the mRNA (post-splicing); useful for infering protein level changes $$$ slightly less costly than DNA sequencing - as the extrons are not sequenced
Restriction fragment length polymorphism (RFLP) PCR, restriction endonuclease digestion, gel electrophoresis useful for finding common base pair changes $$ value of result depends on RFLP data specific to gene, i.e. knowledge about mutations commonly seen in the gene
Amplification-refractory mutation system (ARMS) PCR with mutation-specific primer, gel electrophoresis useful for finding a specific known change $$ primers can be thought of as a hybridization probe; no mutation-specific hybridization (of primer) --> no PCR product
Southern blot gel electrophoresis, hybridization probe with label useful for finding a specific known change, quantifying gene copy number $$$$$ -rarely done
-does not use PCR
-considered the gold standard for clonality[1]
-most labs consider fresh or frozen tissue a requirement[2]

Cytogenetics

A comparison of ISH and karyotyping:

Name of technique Key elements Type of change detected Cost Other
Interphase ISH break apart probe (two colours) probes label two parts of a (normal) gene; the two markers straddle (common) break points gene fragmentation consistent with translocation; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss) $$$$ can detect translocations - without knowing the specific fusion product
Interphase ISH fusion probe (two colours) probes label different genes (that are not adjacent) translocation involving the two genes labeled; one may find: gene duplication (or chromosomal duplication), gene loss (or chromosome loss) $$$$ can detect one specific translocation
Interphase ISH probe (one colour) probe labels one region (gene) gene duplication (or chromosomal duplication), gene loss (or chromosome loss) $$$
Karyotyping metaphase nuclei large scale changes (fusions, deletions, translocations) $$$$ gives the "big picture" view of all the (nuclear) DNA
Metaphase ISH probe (one colour / two colours) probe labels one region (one colour) or probes label two parts of a (normal) gene (two colours) or probes label different genes (two colours) gene duplication, gene loss, translocations $$$$$ rarely done; follows karyotyping to better characterize unusual cases; can be thought of as a karyotype and a simultaneous ISH


Data formats

Human gene naming is provided by the HUGO Gene Nomenclature Committee: https://www.genenames.org/

DNA data repositories

DNA sequence data formats

  • GenBank: human readable, can be processed by computer (fixed width, first 10 characters are an identifier).
    • NCBI Reference Sequence (RefSeq) project provides sequence records and related information.
    • Prefix AC_ in acession number is for genomic data, NM_ is for mRNA.
  • FASTA: Sequence information
    • Header starts with > and is followed by a sequence ID.
    • Sequence lines should wrap always at the same width.
    • Lower-case letters may indicate repetitive regions.
  • FASTQ: Current standard for sequencing data
    • It is essentially FASTA with quality values for the sequence.
    • Quality is on a scale from 0 - 40 and represented by a distinct character.
    • Upper case letters ABCDEFGHI means high quality.
    • Special letters !"#$%&'()*+,-. mean low quality.

Polymerase chain reaction-based techniques

Abbreviated PCR-based techniques
PCR redirects here

General

  • A molecular technique to duplicate DNA (or RNA) molecules ("amplify") and allow the DNA (or RNA) sequence to be determined.

Utility?

  • Detect very small molecular changes - submicroscopic.
    • Changes in sequence - may be as small as one base pair.
  • Used to confirmation chromosomal translocations that are, in clinical practice, usually found with other techniques.

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):[3]
    • 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).[4]
    • 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 formalin fixed paraffin imbedded tissue.
    • RNA is usually extracted with acid guanidium thiocyanate, phenol and choroform.[5]
    • DNA is extracted using phenol and isopropanol.[6]

Other molecular tests

Techniques

  • Southern blot.
    • DNA quantification.

Key elements:

  • Gel electrophoresis.
  • Labeling with hybridization probe.

Cytogenetics

This deals with karyotyping and ISH.

Miscellaneous stuff

World protein databank

The protein databank's goal is to maintain a free and publicly available archive.[7] Ironically, its announcement is in a pay-for-access journal (Nature Structual Biology).[8]

Wnt/beta-catenin pathway

Important in hepatoblastomas.[9]

See also

References

  1. Medeiros, LJ.; Carr, J. (Dec 1999). "Overview of the role of molecular methods in the diagnosis of malignant lymphomas.". Arch Pathol Lab Med 123 (12): 1189-207. doi:10.1043/0003-9985(1999)1231189:OOTROM2.0.CO;2. PMID 10583924.
  2. Reinartz, JJ.; McCormick, SR.; Ikier, DM.; Mellgen, AM.; Bonham, SC.; Strickler, JG.; Mendiola, JR. (Sep 2000). "Immunoglobulin heavy-chain gene rearrangement studies by Southern blot using DNA extracted from formalin-fixed, paraffin-embedded tissue.". Mol Diagn 5 (3): 227-33. doi:10.1054/modi.2000.19808. PMID 11070157.
  3. 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.
  4. URL: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml. Accessed on: 10 May 2011.
  5. 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.
  6. 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.
  7. Worldwide Protein Data Bank. URL: http://www.wwpdb.org/faq.html Accessed on: April 22, 2009.
  8. 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.
  9. 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