This document provides an overview of molecular pathology and various molecular techniques. It discusses the use of nucleic acid testing for clinical purposes such as diagnosis, prognosis, and pharmacogenetics/pharmacogenomics. It describes several molecular techniques including DNA sequencing, PCR, real-time PCR, FISH, microarrays, and their applications. It also discusses DNA/RNA structure, gene expression, mutations, molecular pathology specimen handling, and instrumentation.

1. AKASH MALI ,India
Forensic medicine
Vytautas Magnus University,Lithuania.

2. Molecular Pathology
 Testing of nucleic acids within a clinical context
 Helpful
 Hereditary disorders
 Oncology
 Infectious diseases
•Specific purposes
•Diagnosis
•Prognosis
•Prenatal testing
•Pharmacotherapy
•Pharmacogenetics
•Pharmacogenomics
Molecular pathology employs an ever-expanding array of special techniques to study
nucleic acids, genes, gene products, receptors, signaling pathways, the cell cycle, and
mutations.

4. Watson and Crick
 The structure of DNA was described by British
Scientists Watson and Crick as long double helix
shaped with its sugar phosphate backbone on the
outside and its bases on inside; the two strand of
helix run in opposite direction and are anti-
parallel to each other. The DNA double helix is
stabilized by hydrogen bonds between the bases
 Doctortvrao’s ‘e’ learning series

5. DNA
 A molecule contains two polynucleotide strands that
form an an antiparallel double helix.
 Nucleotides:
 Nitrogenous base (AT GC,U)
 Deoxyribose
 Phosphate

6. DNA makes a Copy of Self
 Replication is the process
where DNA makes a copy
of itself. Why does DNA
need to copy? Simple: Cells
divide for an organism to
grow or reproduce, every
new cell needs a copy of
the DNA or instructions to
know how to be a cell.
DNA replicates right
before a cell divides.

7. DNA – RNA – DNA
a never ending cycle
 RNA has the job of taking
the message from the DNA
to the nucleus to the
ribosome's.
 Transcription - RNA is
made from DNA
 Translation - Proteins are
made from the message on
the RNA
 Doctortvrao’s ‘e’ learning series

8. RNA = Ribonucleic acid.
 RNA is similar to
DNA except:
It has one strand
instead of two
strands. Has uracil
instead of thymine
3.Has Ribose instead of
Deoxyribose

9. Gene Expression
 DNA level expression control
 Transcriptional
 Post-Transcriptional
 Epigenetics
 DNA methylation
 Histone modification

10. Gene Expression
 DNA level expression control
 Transcriptional
 House keeping genes
 Always on
 Transcription factors
 Usually lie upstream in the promoter region
 Enhancer and silencer elements

11. Gene Expression
 Post transcriptional
 Export of mRNA out of nucleus
 Alternative splicing
 mRNA stabilization
 mRNA degradation
 RNA interference or silencing
 miRNA and siRNA

12. What is Gene
 The gene, the basic
units of inheritance;
it is a segment within
a very long strand of
DNA with specific
instruction for the
production of one
specific protein.
Genes located on
chromosome on it's
place or locus.

13. Mutations and Polymorphisms
 Mutation: change in DNA sequence
 Polymorphism: non disease causing change in DNA or
a change found at a frequency of ≥ 1% in population
 When evaluating changes in DNA sequence use
neutral terms: sequence variant, sequence alteration or
allelic variant. There may be:
 Missense, nonsense, deletions, insertions, frame shifts,
duplications, amplifications, trinucleatide repeats.

14. Single Nucleotide Polymorhisms
and Haplotypes
 SNPs are single base differences in the DNA of
individuals
 There are ~10 million SNPs in the human genome
 IMPORTANCE: Pharmacogenetics
 Ex. CYP (cP450)
 Alleles of SNPs that are close together tend to be
inherited together.
 Haplotype: a set of associated SNPs alleles in a region
of a chromosome

15. Overview of Molecular Techniques
and Instrumentation
 Standard or usual specimen flow
 Specimen collection (blood, tissue)
 Nucelic acid isolation (DNA or RNA)
 Nucleic acid quantification (optional)
 Nucleic acid storage
 Nucleic acid amplification (or other)
 Test interpretation
 Quality control

16. Nucleic acid isolation (DNA or RNA)
 Manual vs. automated
 Cell lysis
 Dependent of specimen type, nucleic acid being isolated for,
desired purity and application to be used in
 FFPE yields ~200 pairs
 Purification
 Organic: phenol-chloroform
 Non organic: silica, anion exchange chromatography and
magnetic particles
 DNA or RNA Isolation
 RNA rapidly degrades…

17. Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid Sequence-
Based Amplification
 Signal amplification
 Branching DNA
 Hybrid Capture
 Invader
 FISH
 DNA arrays and chips

18. Gene sequencing
 Determining the exact sequence of the four bases in a
given DNA template
 Two methods
 Maxam-Gilbert
 Chemical degradation
 Sanger
 Chain termination
 Radiolabeled, Dye-prime or Dye-terminator (cycle
sequencing)
 Pyrosequencing
 Sequnces a short length of DNA (~30-60 bases)

19. Applications
of Direct DNA sequences
Clinical condition Gene
HIV drug resistance HIV-protease, RT
Cystic fibrosis CFTR gene
Beta thalassemia Beta globin
Cancer predisposition
• breast BRCA1
•Hereditary non polyposis colon
cancer
TP53
•MEN PTEN Ret proto-oncogene
Congenital hearing loss Connexin 26
HCV genotyping 5’UTR

20. Array-based Comparative Genomic
Hybridization
 Comparative Genomic Hybridization is done in
metaphases in classical cytogenetics (M-CGH)
 Resolution 5 Mb
 Bacterial Artificial Chromosome (BAC) maps the
human genome therefore an Array based-CGH can be
created (A-CGH). Different resolutions up to 32,000
(45 kb)
 cDNA-CGH
 Oligonucleotide-CGH
 Can detect Single Nucleotide Pleomorphisms (SNPs)
[Gene Chip]

22. Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid Sequence-
Based Amplification
 Signal amplification
 Branching DNA
 Hybrid Capture
 Invader
 FISH
 DNA arrays and chips

23. Southern Blot
 Edwin M Southern, 1974
 DNA extracted
 DNA cut into pieces (Restriction Endonucleases)
 Electrophoresis and size separated
 Blot (transferred) to a membrane
 Anealed with labeled (radioactive, fluorescence,
chemiluminescent) probe

24. Southern Blot
working protocol

25. Uses of Southern Blotting
 Southern blots are used in gene discovery and mapping,
evolution and development studies, diagnostics and
forensics.
In regards to genetically modified organisms, Southern
blotting is used as a definitive test to ensure that a
particular section of DNA of known genetic sequence has
been successfully incorporated into the genome of the host
organism.
 Used in prognosis of cancer and in prenatal diagnosis of
genetic diseases

26. Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid Sequence-
Based Amplification
 Signal amplification
 Branching DNA
 Hybrid Capture
 Invader
 FISH
 DNA arrays and chips

27. PCR
 Kary B. Mullis 1983
 Target amplification
 Single oligonucletide
 Multiplexed
 Mimics the natural process of DNA replication, therefore,
requires:
 DNA template, DNA polymerase, dNTPs, buffer, Mg++, two
primers to flag the target sequence
 Thermal cycler
 Denaturation ~95°C
 Annealing ~45-60°C
 Extension ~72°C

28. PCR
 Denaturation
 Breaks the hydrogen bonds between the ds-DNA
 Anealing
 Binding to oligonucleotide sequence (probe)
 Extension
 DNA polymerase (heat stable, Taq [Thermophilus
aquaticus]) replicates the selected DNA sequence
 Xn = X0 × (1 + E)n E= 0 - 1

29. RT-PCR
 To detect or quantify RNA transcripts or viral RNA
 RNA is converted to DNA
 Reverse transcriptase (Avian Myeloblastosis Virus and
Moloney Murine Leukemia virus)
 Isothermal reaction with primers: oligo dT, random
hexamer primers, or target specific primers
 One step vs. two steps

32. PCR or RT-PCR
 Product analysis / detection
 Real Time
 Hybridization
 Membrane bound
 Reverse line blots
 Liquid Bead Array with Flow Cytometry
 Electrophoresis
 Agarose
 Capillary
 Cycle sequencer

34. Multiplexed – PCR and ELISA
Protein Expression Profiling
Cancer Markers
Cardiac Markers
Cellular Signaling
Cytokines, Chemokines, and Growth
Factors
Endocrine
Isotyping
Matrix Metalloproteinases
Metabolic Markers
Neurobiology
Transcription Factors/Nuclear Receptors
Genomic Research
FlexmiR® v2 Custom microRNA Assay
FlexmiR microRNA Panels
Gene Expression Profiling
Genotyping
Genetic Disease
Cystic Fibrosis
Cytochrome p450
Immunodiagnostics
Allergy Testing
Autoimmune Disease
HLA Testing
Infectious Disease
Vaccine Testing
Newborn Screening
Biodefense/Environmental

38. Real Time - PCR
 Amplifies and detects PCR product fluorescently in
each well of PCR plate
 Don’t have to run gel afterwards
 Use for endpoint detection
 Examples
 Fast PCR screening without gels
 Locate clone or mutant of interest
 Genotyping SNPs
 Genotype individuals using allele specific primers

40. PCR
Advantages Disadvantages
 Sensitivity
 Specificity
 Speed
 Versatility
 Automated
 No need for intact DNA/RNA
 Target sequence needs to be
known
 Target needs to be conserved
among individuals
(polymorphisms)
 Oligonucleotide length
 Can fail in the detection of
chromosomal abnormalities like
translocations, inversions, large
addition or deletions
 Contamination (F+)

41. Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid Sequence-
Based Amplification
 Signal amplification
 Branching DNA
 Hybrid Capture
 Invader
 FISH
 DNA arrays and chips

42. Branched DNA applications
 Detection HIV, HBV,
and HCV
 Measures viral loads
 Less sensitive than
PCR
 Doctortvrao’s ‘e’ learning series

43. Hybrid Capture
 Qiagen
 Signal amplification technique
 Denaturated DNA gets hybridized to complimentary
unlabeled RNA sequences (if DNA sequence is present)
 Antibody bound to the well is attracted to RNA:DNA
hybrids
 A second conjugated anti RNA:DNA hybrid antibody is
added
 Chemiluminescent signal is generated in proportion of
target DNA present

46. Applications in Anatomic Pathology
1. Anatomic Pathology Testing to Detect or Characterize
Neoplasia.
2. Molecular Anatomic Testing for Targeted Therapies.
3. Anatomic Pathology Testing for Infectious Agents.

47. 1. P.T. Cagle, T.C. Alan (Eds.), Basic Concepts of Molecular Pathology. Molecular
Pathology Library, vol. 2, Springer Science and Business Media, 2009.