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Core Curriculum in Molecular Biology Copyright 2010 American Society of Cytopathology
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American Society of Cytopathology
Core Curriculum in Molecular Biology
Chapter 3
Molecular Techniques
Molecular Methods in the Diagnostic
Laboratory
Marilee Means, PhD, SCT(ASCP)
University of Kansas Medical Center
Kansas City, Kansas
Copyright 2010 American Society of Cytopathology
Methods Used
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PCR with a variety of analysis of products
Direct Hybridization
Southern Blot
Northern Blot
Cytogenetics with FISH
ISH
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Examples of Diseases
Detected
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PCR – Follicular lymphoma
Direct hybridization – detection of pathogens
Southern blot – Burkitt lymphoma
Northern blot – neuroblastoma
FISH – bladder carcinoma
ISH – HER2/neu/erb-B2 in breast CA
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PCR – polymerase chain reaction
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Advantages – specificity, sensitivity, speed
Can use very small amount of DNA
Many types specimens (blood, CSF, etc)
Disadvantages – Must know DNA
sequence to design primers for PCR
• Extreme sensitivity, PCR contamination,
specificity (too low a temp can anneal
wrong DNA), cost, targeted nature rather
than swab or karyotype
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Copyright 2010 American Society of Cytopathology
-http://www.genome.gov/10000207#1
Animation of PCR cycles
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• www.dnalc.org/ddnalc/resources/pcr.html
• Note that the cycling can produce over a
million copies of the DNA segment of
interest in a little over 30 cycles.
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Analysis of PCR Products
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• Gel electrophoresis – size, pore size, net
charge, structure of molecule, etc.
• Restriction fragment length polymorphism
–if disease state alters restriction enzyme
cut site, then lengths vary from normal
• Single stranded conformational
polymorphism analysis – denature DNA to
single strand which folds differently than
normal
Copyright 2010 American Society of Cytopathology
Gel electrophoresis animation
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• http://www.dnalc.org/ddnalc/resources/ele
ctrophoresis.html
Copyright 2010 American Society of Cytopathology
Analysis of PCR Products
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• Hybridization – for detection of genetic
diseases, cancers, infectious organisms
• Sequence Analysis – for confirming known
mutations or searching for unknown
mutations in small genes
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Diagnostic Applications of PCR
• Gene mutation detection – Can detection point
mutations or single nucleotide polymorphisms
(genetic, cancer, infectious)
• Linkage Analysis – if the chromosomal location
is known but the gene or mutation is not yet
identified (Variable number of tandem repeats –
VNTR – long chains of repeats analyzed by
southern blot OR Short tandem repeats –
analyzed by PCR)
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Diagnostic Applications of PCR
• Microbiology-Enhance traditional culture and
serology tests, usually targeted for a specific
organism
• PCR and hybridization are the usual uses in
microbioloby
• PCR is rapid, sensitive, and specific, doesn’t
require fresh material, good for slow or difficult
to grow organisms
• A few TB organisms can be ID in hours rather
than weeks of culture (>10ⁿ/ml) (4)
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Microbiologic Applications
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• Viruses such as herpes, HIV, hepatitis B
and C
• Outbreak organism tracking, such as
investigation of source of E. coli in spinach
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Direct Hybridization Methods
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• Uses a probe complementary to DNA to
be detected
• Used for pathogen detection
• Must know the target DNA sequence
• May be stringent (only one unique
sequence from one organism) or several
sequences in gene families
• Sensitivity depends on type of label and
stringency
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Southern Blot Analysis
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• Genetic diseases, antigen receptor gene
rearrangements, and viral infections
• Reliable, specific, accurate, method of
choice for many clinical applications
• Disadvantages from PCR – requires large
amounts of intact genomic DNA, cyto and
histology may not yield enough intact
DNA, no amplification step, time and labor
intensive
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Southern Blot animation
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• http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5
35::535::/sites/dl/free/0072437316/120078
/bio_g.swf::Southern%20Blot
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Northern Blot analysis
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Time and labor intensive
Not frequently used in clinical lab
Used to analyze mRNA
Steps are similar to Southern blot
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Cytogenetics
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• Study of chromosomes to detect changes
in number and structure
• Analyzes chromosomes by size, banding
pattern (karyotyping)
• Molecular cytogenetics combines
cytogenetics with molecular techniques to
resolve problems with growth of live cells,
detection of small abnormalities, in vivo
growth patterns may skew analysis
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Fluorescence in Situ Hybridization
FISH
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• Can analyze DNA within the cell and
correlate with histology and cytology
• Used to determine number of copies and
location of a specific DNA sequence in the
cell
• Doesn’t need dividing cells, therefore no
cell culture = reduced turnaround time
• Wide range of specimen types including
fixed tissues and cells
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FISH
http://www.genome.gov/12514471
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Abnormal
Note too many reds,
blues, and greens and
loss of golds
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Normal
Note two each of
red, blue, green, and
gold
In situ hybridization
ISH
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• Similar to FISH but the probe is identified
microscopically (without fluorescent probe)
• Less sensitive than amplification methods
• Used to locate viruses within cell such as
HPV, EBV, hepatitis in a variety of
malignancies
• Detect oncogene expression such as MYC
oncogene in multiple myeloma and
HER2/neu/erb-B2 in breast CA
Copyright 2010 American Society of Cytopathology
Summary
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• PCR, Direct hybridization, Southern blot,
Northern blot, cytogenetics, FISH, and ISH
are more and more commonly used to aid
in the detection and treatment of disease
• Many of these techniques can be used as
an adjunct to cytopathology
Copyright 2010 American Society of Cytopathology