Molecular pathology

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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.


Molecular pathology can be divided as follows:

based techniques

Tabular comparisons


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]


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:

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


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


  • 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.


  • 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


  • Southern blot.
    • DNA quantification.

Key elements:

  • Gel electrophoresis.
  • Labeling with hybridization probe.


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


  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: 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: 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