Download DNA, RNA and Protein

DNA, RNA and Protein: A Basic Science
Tutorial for SLPs
Rebecca Bartlett, MA, Marie Jetté, MS, Suzanne
King, MS, Allison Schaser, MS, Susan Thibeault, PhD
Introduction: Diane M. Bless, PhD
Raise your hand if you…
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•
•
•
•
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Are an SLP
Are an audiologist
Work clinically
Have research experience
Work in a laboratory
Read articles which include molecular
biology concepts
Our Purpose
Don’t be afraid!
Bench
Clinic
Community
Central Dogma of Molecular Biology:
DNA  RNA  Protein
Central Dogma of Molecular Biology
• Techniques are
available to
measure
– DNA
– RNA
– Protein
Goal
1. Provide necessary knowledge to foster
comprehension of the research literature
2. Provide necessary knowledge to
understand the translation of the
methodology to clinical applications
Format
• DNA – Marie Jette
• RNA – Rebecca Bartlett
• Protein – Suzanne King and Allison
Schaser
DNA
Transcription
RNA
Translation
Protein
Let’s start with a 5 question quiz!
DNA Quiz
You have just landed your first job as a speech
pathologist and now have the opportunity to
build the house of your dreams. If this scenario
described the central dogma of biology, which
represents DNA?
A. All of your ideas for the house
B. Blueprints RNA
C. Building supplies proteins
DNA
Which three people were
awarded the Nobel Prize for the
discovery of the structure of
DNA: the double helix?
A.
B.
C.
D.
James Watson, Francis Crick, and Rosalind Franklin
James Watson, Francis Crick, and Albert Einstein
James Watson, Francis Crick, and Charles Darwin
James Watson, Francis Crick, and Maurice Wilkins
Our individual DNA makes us
unique, but it also makes us
human. What percentage of our
DNA do humans have in
common with our closest primate
cousin, the chimpanzee?
A. Over 60%
B. Over 89%
C. Over 98%
Where would you expect to find
genetic information describing
the characteristics of a Dodo?
A. In virtually every cell
in its body.
B. In the ribosomes.
C. In the primary
spermatocytes.
D. In its feathers.
True or False: DNA is a
chromosomal blueprint that
does not change throughout a
lifespan.
A. True
B. False
Deoxyribonucleic Acid (DNA)
DNA
Every cell in the body contains a complete
copy of DNA (except red blood cells)
Chromosome? Gene? DNA?
How are genes named?
• HUGO Gene Nomenclature Committee
(HGNC) has assigned unique gene
symbols and names to more than 32,000
human genes
• Genes that make you go “hmmmm…”
– lunatic fringe
– head case
– one-eyed pinhead
– sonic hedgehog
www.pbs.org/wnet/dna/episode1/index.html
Polymerase Chain Reaction
(PCR) for making more DNA
http://missinglink.ucsf.edu/lm/molecularmethods/images/clip_image003.gif
Kary Mullis and PCR
PCR: An Analogy
Photocopier Analogy
Photocopier items
The book
PCR components
The entire genome (DNA
template)
The page being copied
A portion of the genome
(fragment) we are interested
in
Highlighter
Primers that “mark” the
specific fragment
Steps for Performing PCR
1. Extract DNA from cells
2. Combine DNA, primers, and other
molecules in a reaction mixture
3. Put reaction mixture in a heating and
cooling machine (aka, thermocycler)
–
Cycles of heating and cooling result in exponential
growth of DNA fragment of interest
4. Verify amplification with gel
electrophoresis
What can we learn from PCR?
• Detecting bacteria and viruses in samples
from sick people, sometimes before they
develop symptoms
• Diagnosing genetic diseases and mutation
• Paternity testing
• Forensics
PCR Example
Clinical question: Is a mutation in the gene
FOXP2 related to autism?
Preliminary goal: Use PCR to detect and amplify
FOXP2 gene in whole blood from families with
autism.
Wassink et al. (2002). Evaluation of FOXP2 as an autism susceptibility gene. American Journal of Medical
Genetics, 114(5), 566-569.
PCR Example
Methods:
1.Extract DNA from whole blood
2.Amplify FOXP2 gene mutation using genespecific primers
3.Verify amplification
Measure = Presence of FOXP2 gene
PCR Example
Conclusions:
It is unlikely that FOXP2 contributes
significantly to the predisposition to
develop autism in the study sample.
Significance:
Autism is a heterogeneous diagnosis with no
single gene responsible for its features.
PCR
Advantages:
• Invaluable technique for
measuring gene expression
Limitations:
• Poor precision
• Results are non-quantitative
Putting it all together…
• To determine strain of HPV associated
with recurrent respiratory papillomatosis
• To detect presence of bacteria in laryngeal
biopsies
DNA
Transcription
RNA
Translation
Protein
RNA Quiz
You have just landed your first job as a speech
pathologist and now have the opportunity to
build the house of your dreams. If this scenario
described the central dogma of biology, what
represents RNA?
A. All of your ideas for the house DNA
B. Blueprints RNA
C. Building supplies proteins
Which of the following is NOT a
source of RNA?
A.
B.
C.
D.
Using an RNA-based test,
scientists were able to classify
this ambiguous lesion as:
A.Reinke’s edema
C. Cyst
B. Polyp
D. Granuloma
messengerRNA (mRNA)
mRNA
• sent from DNA in nucleus  cytoplasm
• tells the cell which proteins to make
Sources of mRNA
mRNA Transcription
1. DNA strands separate
2. RNA polymerase
transcribes DNA into a
strand of mRNA
Adenine – Uracil
Cytosine - Guanine
3. mRNA strand leaves
nucleus
Video-Transcription
www.pbs.org/wnet/dna/episode1/index.html
mRNA Transcription
Cell nucleus
mRNA strand
mRNA Assays
• Quantitative PCR
• Gene Microarray
Quantitative Polymerase Chain
Reaction (qPCR)
product
PCR
qPCR
cycles
PCR- Product detection after ~40 cycles
qPCR/RT-PCR- Product detection after each cycle
qPCR Example
Clinical question: Can genetic markers predict which
patients with leukoplakia will develop cancer?
Preliminary goal: use qPCR to identify genetic
expression profiles to distinguish leukoplakia lesions
Bartlett RS, Heckman WW, Isenberg J, Thibeault SL, Dailey SH, Genetic characterization of vocal fold lesions: Leukoplakia and
carcinoma. Laryngoscope, (in press).
qPCR Example
Methods:
1. Leukoplakia biopsies:
1. Non-dysplasia  2. Dysplasia  3. Cancer
2. RNA extraction
3. 96 gene Human Cancer Pathway arrays
qPCR Array
qPCR Example
Conclusions:
• Genes distinguished the groups
Significance:
• With a larger study, it may be possible to
identify predictive gene markers for vocal
fold carcinoma
Bartlett RS, Heckman WW, Isenberg J, Thibeault SL, Dailey SH, Genetic characterization of vocal fold lesions: Leukoplakia and
carcinoma. Laryngoscope, (in press).
PCR
qPCR
• Semi quantitative
• Quantitative
• Measures product
once
• Measures product
after each cycle
qPCR
Advantages:
• High sensitivity
• No post-PCR steps are needed
• Relatively high throughput
Limitations:
• Requires specialized
equipment
• Expensive
Microarray
•
Can assess up to 30,000 genes
simultaneously
Applications:
• Early disease detection
• Newborn disease screening
• Drug discovery
• Detection of bacteria/viruses in water, soil,
etc
Microarray Example
Clinical question: Can genetic analysis assist
in differential diagnosis of visually similar vocal
fold lesions?
Preliminary goal: Use microarray to compare
gene expression of polyp and Reinke’s edema
tissue
Duflo, Thibeault, Li, Smith, Schade, Hess. Differential gene expression profiling of vocal fold polyps and reinke’s edema by
complementary DNA microarray. Annals of Otology, Rhinology and Laryngology, 2006.
Microarray Example
Methods:
1. RE and polyp lesions were
biopsied
2. RNA extraction
3. Samples screened for 8,745 genes
 Obtain biopsy tissue
 Extract mRNA
 Label samples with red
or green tags
 Apply samples to chip
and capture fluorescence
Microarray chip
= Reinke’s edema
= Polyp
= Both samples
= Neither sample
1 spot = 1 gene
Microarray Example
Conclusions:
Reinke’s edema
• 65 genes differentiated RE and polyps
Significance:
• The significant genes could classify
ambiguous lesions
Duflo, Thibeault, Li, Smith, Schade, Hess. Differential gene expression profiling of vocal fold polyps and reinke’s edema by
complementary DNA microarray. Annals of Otology, Rhinology and Laryngology, 2006.
Microarray
Advantages:
• High throughput***
Limitations:
• Costly equipment
• Less accurate than qPCR for quantifying
expression differences
Putting it all together
• To screen a genetic sample from a
newborn for 10,000 genes associated with
congenital disorders, use: MICROARRAY
• A child in the SLP clinic has syndromic
facial features but no diagnosis. To test
their genetic sample for 10 genes
associated with the most common
syndromes, use: qPCR
BREAK TIME!
DNA
Transcription
RNA
Translation
Protein
Protein Quiz
You have just landed your first job as a speech
pathologist and now have the opportunity to
build the house of your dreams. If this scenario
described the central dogma of biology, which
represents protein?
A. All of your ideas for the house
B. Blueprints RNA
C. Building supplies proteins
DNA
Which of the following can
cause disease?
a.A protein that contains an incorrect amino
acid
b.A protein that is incorrectly folded
c.A protein that is missing from the body
d.All of the above
Which one of these does NOT
contain protein?
Trick Question!
These are all
sources of protein 
Proteins make up more than ½
of your body’s non-water mass?
TRUE
FALSE
Sources of Protein
http://web.jjay.cuny.edu/~acarpi/NSC/images/cell.gif
What are proteins?
http://mrswolfgang.wikispaces.com/file/view/myprotein%3D).png/61409292/myprotein%3D).png
How are proteins made?
How are proteins made?
www.pbs.org/wnet/dna/episode1/index.html
Protein’s Structural Complexity
What does protein do?
Structure
Cell
Signaling
Gene
Expression
Movement
Bind
Catalyze
Protein Assays
• ELISA
• Immunohistochemistry
• SDS-PAGE & Western Blot
Basic Concept of Protein Assays
Antibodies & Antigens
Immunoassays rely on ability of an individual
antibody site to bind\react with only one
antigen (i.e. protein, virus, bacteria)
– Lock and Key Concept
Antigen
A substance that stimulates the production of
antibodies:
– Foreign substances
– Proteins
– Bacteria
– Viruses
Antibody
A protein that is
produced by the body
in response to a
“invading” (foreign)
substance.
Antibody & Antigens
Lock and Key Concept:
• Antigens can bind in pockets or grooves
of antibodies
Protein Assays
• ELISA
• Immunohistochemistry
• SDS-PAGE & Western Blot
Enzyme Linked ImmunoSorbant
Assay (ELISA)
Technique:
1. Detect antigens or
antibodies
2. Measure quantity in a
sample
Clinical Applications:
1. Pregnancy tests
2. HIV screenings
3. Detect Food Allergies
4. Reflux diagnosis
ELISA Example
• Clinical Question:
– Can Laryngopharyngeal
Reflux (LPR) be accurately
diagnosed using a pepsin
ELISA?
• Preliminary Goal:
– To establish a subjective
measure for diagnosing
patients with LPR?
Knight J, Lively MO, Johnston N, Dettmar PW, Koufman JA. (2005). Sensitive pepsin immunoassay for detection of
laryngopharyngeal reflux. Laryngoscope,115(8) 1473-8.
Methods
• Designed a non-invasive “Spit in a cup test”
– Pepsin: enzyme in digestive system that degrades
proteins
• Compared pepsin levels in throat sputum to 24 hr
double-probe pH monitor
– Tested 23 LPR patients
Knight J, Lively MO, Johnston N, Dettmar PW, Koufman JA. (2005). Sensitive pepsin immunoassay for detection of
laryngopharyngeal reflux. Laryngoscope,115(8) 1473-8.
Pepsin “Sandwich” ELISA
Enzyme Label
Detection
Antibody
Pepsin
Capture
Antibody
96 well plate
Uses an indirect method (multiple antibodies) to detect and
measure pepsin levels from throat sputum samples
ELISA Technique
ELISA Technique
ELISA Example
Conclusion: pepsin ELISA
showed high sensitivity (100%)
and specificity (89%) for
pharyngeal reflux events
Significance: pepsin ELISA can
be used clinically to accurately
detect LPR
Knight J, Lively MO, Johnston N, Dettmar PW, Koufman JA. (2005). Sensitive pepsin immunoassay for detection of
laryngopharyngeal reflux. Laryngoscope,115(8) 1473-8.
Immunohistochemistry
Technique:
1. Detect antigen in a tissue
slice
2. Maintain morphology of
the sample
3. Measure quantity in a
sample
Clinical Applications:
1. Post-Mortem Autopsy
Diagnosis
2. Tumor Typing in Cancer
Diagnosis
Immunohistochemistry
Techniques
Immunohistochemistry
Techniques
microscope-microscope.org
Immunohistochemistry Example
• Clinical Question:
– Is cellular stress an early event in
Alzheimer’s Disease?
• Preliminary goals:
– Look at differences in cellular stress in
brains of elderly individuals
– Correlate levels of cellular stress with
functional deficits
Schipper, H. M., Bennett, D. A., Liberman, A., Bienias, J. L., Schneider, J. A., Kelly, J., et al. (2006). Glial heme
oxygenase-1 expression in alzheimer disease and mild cognitive impairment. Neurobiology of Aging, 27(2), 252261.
Immunohistochemistry Example
• Methods:
– Used IHC techniques
to stain for marker of
cellular stress in post
mortem tissue
Schipper, H. M., Bennett, D. A., Liberman, A., Bienias, J. L., Schneider, J. A., Kelly, J., et al. (2006). Glial heme oxygenase-1
expression in alzheimer disease and mild cognitive impairment. Neurobiology of Aging, 27(2), 252-261.
Immunohistochemistry Example
No Cognitive
Impairment (NCI)
Mild Cognitive
Impairment (MCI)
Alzheimer’s
Disease (AD)
Schipper, H. M., Bennett, D. A., Liberman, A., Bienias, J. L., Schneider, J. A., Kelly, J., et al. (2006). Glial heme
oxygenase-1 expression in alzheimer disease and mild cognitive impairment. Neurobiology of Aging, 27(2), 252261.
Immunohistochemistry Example
• Conclusion:
– Individuals with NCI had significantly
less cellular stress than individuals with
MCI and AD, no difference between MCI
and AD
– Levels of cellular stress were correlated
with measures of global functioning and
memory
Schipper, H. M., Bennett, D. A., Liberman, A., Bienias, J. L., Schneider, J. A., Kelly, J., et al. (2006). Glial heme
oxygenase-1 expression in alzheimer disease and mild cognitive impairment. Neurobiology of Aging, 27(2), 252261.
Immunohistochemistry Example
• Significance:
– Cellular stress is a
early event in the
process of AD and is
linked to functional
deficits
– Mechanisms to
combat cellular stress
should be employed
early in the disease
process.
Schipper, H. M., Bennett, D. A., Liberman, A., Bienias, J. L., Schneider, J. A., Kelly, J., et al. (2006). Glial heme
oxygenase-1 expression in alzheimer disease and mild cognitive impairment. Neurobiology of Aging, 27(2), 252261.
Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) & Western Blot (WB)
Technique:
1. Separate protein based on
molecular weight
2. Use antibodies to determine
specific proteins in sample
3. Measure quantity in a sample
Clinical Applications:
1. Confirmatory tests for different
diseases: Ex: HIV, Lyme
Disease, Hepatitis B
SDS-PAGE
+
−
lanes
a
b
c
largest
a
b
c
smallest
SDS-PAGE/WB Example
• Clinical Question:
– Are there age related changes in the
muscles involved in swallowing that
could explain functional changes
seen in elderly swallow?
• Preliminary goals:
– Examine age-related differences in
muscle fiber types in tongue muscle
using a rat model
Schaser, A. J., Wang, H., Volz, L. M., & Connor, N. P. (2011). Biochemistry of the anterior, medial, and posterior
genioglossus in the aged rat. Dysphagia, 26(3), 256-263.
SDS-PAGE/WB Example
• Methods
– Extract tongue
muscle from elderly
rat
– Use protein
analysis to
measure change in
muscle fiber types
with age
Schaser, A. J., Wang, H., Volz, L. M., & Connor, N. P. (2011). Biochemistry of the anterior, medial, and posterior genioglossus in
the aged rat. Dysphagia, 26(3), 256-263.
Muscle Fiber Proteins
SDS-PAGE/WB Example
• Conclusions:
– Switch to more slowly
contracting fiber types
with age
• Significance:
– Change to more slowly
contracting fiber types
could explain overall
slowing of the swallow
seen with age
Schaser, A. J., Wang, H., Volz, L. M., & Connor, N. P. (2011). Biochemistry of the anterior, medial, and posterior
genioglossus in the aged rat. Dysphagia, 26(3), 256-263.
Advantages & Disadvantages
Advantages:
1.
2.
3.
ELISA: simple, cost-effective and widely
available
IHC: Allows you to maintain structure of tissue
and look at changes in morphology
SDS-PAGE & Western Blot: commonly used
technique throughout the literature, allows for
comparison to previous work
Disadvantages:
1.
2.
3.
Antibodies are expensive
Antibodies need careful controls to make sure
they are working properly
IHC & Western Blot are semi-quantitative
methods
Bench
Clinic
Community
We hope we were able to…
• Provide necessary knowledge to foster
comprehension of the research literature
• Provide necessary knowledge to
understand the translation of the
methodology to clinical applications
Acknowledgements
Diane Bless, PhD
Susan Thibeault, PhD
Nadine Connor, PhD
Thibeault lab
NIH NIDCD R01 DC4336
R01 DC9600
R01 DC008149
T32 DC009401
Connor lab
Questions?
Thank you