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.
3.
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]
21.
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
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
30.
31.
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
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
39.
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
44.
45.
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.
Editor's Notes
ONE STEP: RT + DNA polymerase OR rTth (Thermus themophilus) that works as an RT and DNA polymerase
Hybridization probe: Two separate, single-labeled probes anneal to target bringing donor and reporter into proximity
Dual-labeled hydrolysis probe: When the reporter and quencher fluorophores are in proximity on the probe – there is no signal. Once the 5’nuclease activity of the DNA polymerase hydrolyses the end nucleotides from the probe where the reporter is. The reporter emits a signal.
Minor groove binding probes (MGB): like hydrolysis probes, technique to stabilize shorter probes.
Molecular beacon probes: Annealing to the target after denaturation allows the reporter fluorophore to escape the quenching effect, therefore giving a signal