Molecular Biology in Medicine Flashcards
What are the 4 Methods to analyze DNA?*
- cytogenetics/karyotyping
- RFLP
- Restriction enzymes
- Southern Blotting
What are the 2 Methods to analyze RNA?*
- Northern Blotting
- RPA
What are the 3 Methods to anaylze Proteins?*
- Western Blotting
- IP & co-IP
- ELISA
This is an example of what molecular biology method?
Cytogenetics/Karyotyping
- Early method of genetic testing
How can we identify single nucleotide pair mutations?
Restriction Fragment Length Polymorphism (RFLP)
- uses specific restriction enzymes (endonucleases)
- detects difference in homologous DNA sequences
- presence of fragments of different lengths after digestion
- specific to single clone/restriction enzyme combination
How do restriction enzymes/digestion function?
- Sequence recognition
- Cut DNA at specific site
Restriction enzymes = important for RFLP
Restriction enzymes + Klenow fragments = important for cloning
- DNA ligase
- Complementary “sticky” overhands of the same type (5’ or 3’) OR blunt ends = can be ligated together
- Klenow fragment of DNA polymerase
- Overhangs –> blunt ends
Example of a restriction enzyme: EcoR1 - 5’ overhangs
Recognition sequence/ Cut
5’GAATTC / 5’G AATTC 3’
3’CTTAAG / 3’CTTAA G 5’
What are the 6 steps in a Southern Blot?
- Detect specific DNA sequence
- Cut DNA w/ restriction enzymes
-
Run agrose gel (electrophoresis)
- separate large charged molecules based on size
- optional: acid treat gel to break up large fragments/enhance transfer
- Transfer to filter
- Hybridize labeled probes
- Detect on film/machine
What is hybridization?
- complementary bases needed
- DNA-DNA, RNA-RNA, or DNA-RNA
- temperature dependent
- probe = reverse complement (antisense) of target
DNA probe + DNA = double strand
RNA probe = single strand
What are the 4 steps of a Northern Blot?
- Run RNA on denaturing agrose gel
- Transfer RNA to membrane
- Hybridize to labeled probe
- Visualize on film/machine
Good for:
- measuring RNA-levels
- sizing full-length RNA
- normalize to “housekeeping genes” (actin or GAPDH)
Be careful:
- sensitive to RNA quality
- easily degraded by environmental ribonucleases
What is the Ribonuclease Protection Assay (RPA) procedure?
- Hybridize target RNA w/ labeled probes
- Digest non-hybridized RNA w/ RNAase
- Run on denaturing polyacrylamide gel
- Detect on film
Note: Can normalize to a “housekeeping” gene for quantitation
Result: Only RNA bound to probe is left behind = bands of digested sample
Good for:
- measuring RNA levels
- evaluating RNA processing
- looking at specific RNA areas
- more forgiving of partially degraded RNA
Bad for:
- full-length analysis
Why use a Western Blot?
- Detects proteins (total, cytoplasmic, nuclear)
- Use polyacrylmide gel ALWAYS
Good for:
- Can quantitate if necessary
- Can control of equal loading
- (by ponceau staining or assay of housekeeping genes)
- Can detect varient forms of proteins
- P, de-P
- Detect protein:protein binding
- coimmunoprecipitation (co-IP)
- IP = precipitating a specific protein out of solution with its antibody
- beads bind Ab-protein complex
- if protein bound to other proteins, will also be IP out of solution (co-IP)
Why use a Dot Blot?
-
Simplification of Southern/Northern/Western blots
- for detection of specific DNA, RNA, or protein macromolecules
- Samples spotted on filter w/ probe (RNA/DNA) or antibody (protein)
- “Spot” allows us to determine presence of specific macromolecule
- intensity of spot = estimation of levels
Other detection methods:
In situ hybridization?
- Detect DNA or RNA sequences in tissue or cells
- Use of labeled nucleic acid probes
Note: Useful in pathology
Other detection methods:
Immunohistochemistry?
- Detect proteins in tissue
- via use of antibodies
Note: Useful in pathology
Other detection methods:
Immunocytochemistry?
- Detect proteins in intact cells (usually with most or all of the extracellular matrix removed)
- via use of antibodies
Note: Useful in pathology
What is Enzyme-Linked Immunosorbent Assay (ELISA)?
- Detects proteins, pathogens, etc
- qual or quantitative
- quick & easy
Sandwich ELISA:
- Plate coated w/ capture Ab
- Sample added
- Detecting Ab added, binds to Ag
- Enzyme-linked 2° Ab added & binds to detecting Ab
- Substrate added, converted by enzyme to detectable form
Why use Chromatin Immunoprecipitation (ChIP)?
- Evaluate proteins bound to DNA
- ex: transcription factors bound to gene promoters
- PCR & sequencing = used to analyze purified DNA in ChIP
-
Gel shift experiments = used to analyze protein/nucleic acids (NA)
- Nucleic acid sequence + protein on polyacrylamide gel
- Bound NA run slower
- Nucleic acid sequence + protein on polyacrylamide gel
Good for:
- Understanding mechanisms of disease
- Understanding normal binding of a sequence
Why use a Polymerase Chain Reaction (PCR)?
-
Requires:
- template DNA
- primers
- polymerase
- nucleotides
- Detects & amplify specific DNA
- product doubles w/ each cycle
Process:
- Denaturation (~94°C) - 5 min
- Annealing (~55°C) - 20-40 cycles
- Elongation (~72°C) - 10 min extension
Be careful:
- Efficiency & specificity of rxn can depend on many factors
- ex: sequence, primer, temp
-
Sensitive to contamination
- use negative control (no template = no contamination)
- UV irradiation needed
- clean materials prior to PCR to avoid cross-linking
- Can introduce mutations into product/new restriction enzyme sites
Note: Must have annealing temp LOWER at first few cycles to allow primer-target annealing
Why use Reverse Transcription PCR (RT-PCR)?
-
Requires:
- RNA
- Primers
- Reverse Transcriptase
- DNA Polymerase
- Buffer Reagents
- Can be 1 step or 2 step
Good for:
- Detecting & quantifying mRNA levels
- Evaluating RNA processing
- Creating cDNA (cloning)
Why use Real Time PCR?
- More accurate quantification of DNA or RNA
- can be used with regular PCR or RT-PCR
- Compare cycle when threshold is detected
- Each earlier cycle = 2 x more starting material
Process:
- In intact probes, reporter fluorescence is quenched
- Probes & complementary DNA strand = hybridized and reporter fluorescence is still quenched
- During PCR, the probe is degraded by the Taq polymerase
- fluorescent reporter released
Why use a Microarray?
-
Measure gene expression
- mRNA –> cDNA (quantified)
- more RNA = more cDNA = more bound to chip/filter
- Colors:
- Green = gene more expressed in control
- Red = gene more expressed in sample (tumor)
- Yellow = equal expression in both
- Black = no expression/unknown
-
SNP arrays
- detect presence/absence of varients of large # of SNPs
-
Protein arrays
- can quantify gene expression at protein level
Be careful:
- Not used for RNA processing since cDNA only has expressed exons
What are the steps for DNA Cloning?
-
Make cDNA from (fully mature) mRNA
- Use reverse transcriptase
-
Digest cDNA & vector (plasmid)
- expression vector
-
Ligate cDNA fragment into vector
- intro-dependence problem
- need natural intronless RNA help
Genomic library
- collection of total genomic DNA from a single organism
Why use a Plasmid/Expression Vector?
- Can put cDNA into a plasmid/expression vector
- will see the expression/effect of DNA
- Allows for transfection
- introduction of exogernous DNA into cells
What are the 2 types of Transfection?
-
Transient (short-term)
- extrachromosomal
-
Stable (long-term)
- integrates into genome (selection + screening)
- ex: homologous recombination = exogenous DNA replaces homologous endogenous DNA (wild-type)
What is Sanger Method?
-
Sequencing of DNA
- addition of terminating base
- Requires:
- Primer & DNA template
- DNA polymerase
- ddNTPs w/ fluorochromes
- dNTPs (dATP, dCTP, dGTP, dTTP)
Good for:
- detecting heterozygous point mutations
- N = could be mutation or far from primer
Be careful:
- Huntington’s CAG repeat
What is a Single Nucleotide Polymorphisms?
-
Change of a nucleotide at a single base-pair on DNA
-
Other detections:
- Indels (insertions/deletion of bp)
-
Other detections:
- Also be detected by Microarrays
Strategies for Gene Therapy?
-
Loss of function
- restore function
-
Gain of function
- eliminate abnormal function = harder to do
Methods:
- expression vectors
- siRNA, shRNA
- CRISPR (can make changes to a genetic sequence)
- RNA processing therapeutics
Delivery:
- Viruses
- Nanoparticles
Problems:
- Efficacy vs Specificity
- minimize side effects
-
Sequencing of DNA
- addition of terminating base
- Requires:
- Primer & DNA template
- DNA polymerase
- ddNTPs w/ fluorochromes
- dNTPs (dATP, dCTP, dGTP, dTTP)
Good for:
- detecting heterozygous point mutations
- N = could be mutation or far from primer
Be careful:
- Huntington’s CAG repeat
What is Sanger Method?
-
Loss of function
- restore function
-
Gain of function
- eliminate abnormal function = harder to do
Methods:
- expression vectors
- siRNA, shRNA
- CRISPR (can make changes to a genetic sequence)
- RNA processing therapeutics
Delivery:
- Viruses
- Nanoparticles
Problems:
- Efficacy vs Specificity
- minimize side effects
Strategies for Gene Therapy?
