Molecular Biology in Medicine Flashcards by Jasmine Santos

What are the 4 Methods to analyze DNA?*

cytogenetics/karyotyping
RFLP
Restriction enzymes
Southern Blotting

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What are the 2 Methods to analyze RNA?*

Northern Blotting
RPA

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What are the 3 Methods to anaylze Proteins?*

Western Blotting
IP & co-IP
ELISA

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This is an example of what molecular biology method?

Cytogenetics/Karyotyping

Early method of genetic testing

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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

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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’

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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

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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

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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

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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

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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)

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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

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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

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Other detection methods:

Immunohistochemistry?

Detect proteins in tissue
- via use of antibodies

Note: Useful in pathology

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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

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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

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Why use Chromatin Immunoprecipitation (ChIP)?

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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

Good for:

Understanding mechanisms of disease
Understanding normal binding of a sequence

Why use a Polymerase Chain Reaction (PCR)?

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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)?

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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?

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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?

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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?

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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

What are Reporter Systems?

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Regulatory sequence to be studied
1. ex: gene promoter
Reporter gene
1. encoding GFP OR Luciferase
DNA –> mRNA –> Reporter Protein
1. amount is easily measured via fluorescence

Good for:

Identifying if gene promoter has a mutation
Finding out if promoter affect expression
Will drug affect gene expression

Why use a Plasmid/Expression Vector?

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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?

1. **Transient** (short-term) * _extrachromosomal_ 2. **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 Next Generation Sequencing?

* Massive _parallel sequencing_! * Billions of sequence reads in a single run * ChiP with nucleotides (complimentary sequence) * DNA processing & binding * Amplification & analysis 3 Main Steps: 1. Amplify 2. Sequence 3. Analyze

What is a Single Nucleotide Polymorphisms?

* **Change** of a nucleotide at a **single base-pair on DNA** * _Other detections_: * Indels (insertions/deletion of bp) * **Also** be detected by **Microarrays**

Why use Circulating DNA Diagnostics?

* **Circulating tumor DNA** (ctDNA) * Blood sample contains DNA from all over tumor, not just one section * promising non-invasive biomarker for cancer burden * cancer personalized profiling via **CAPP-Seq** * economical & ultrasensitive ways to quant ctDNA * Used for **diagnosis & monitoring** of _other disorders_ * ex: diabetes + heart disease * **Liquid biopsies**

Types of DNA biomarker detection?\*

1. Direct to consumer * **SNP analysis** * 23andMe 2. Circulating DNA diagnostics * blood sample for **ctDNA** * **non-innvasive** tumor/disease detection/progression 3. Cologuard * To help detect colon cancer * find _elevated levels of altered DNA_ (amplification & detection of methylated target DNA) * find _elevated levels of hemoglobin_ (ELISA) in stool 4. QuARTS * _Detection of methylated versions of gene_ sequence via different primers (similar to Real Time PCR) * **Bisulfite tx** causes **C --\> U (primer read A)**, but **if C is methylated** it will not change **(primer reads G)** * Good for finding _point mutations_ via sequencing 5. MediMap * **pharmacogenomics (PGx) test** on newborns * looks for _variations in 7 genes for drug proccessing issues_

Strategies for Gene Therapy?

1. **Loss of function** * restore function 2. **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

Types of Cancer Immunotherapy

1. **Passive Immunotherapy** * _Antibodies fight Cancer Antigens_ * Ab made outside the body & administered to patient * Drugs: -ib = inhibitors * Drugs: -mab = monoclonal Ab 2. **Active Immunotherapy** * _Engineer patient's immune cells_ to fight their cancer * triggers pt's immune sys to respond to disease * vaccines w/ pt's cancer cells co-cultured * vector-based cancer vaccine = introduce cancer-specific proteins to pt * _Limitations_ * tumor cells/Ags mutate * low response rate * manufacturing challenges * cancer vaccines = poorly immunogenic --\> can be toxic * development of autoimmune disease 3. **Activate patient native immune system** to fight the cancer * **"Release the brakes"** * Block the "programmed death 1 (PD-1)" pathway in cell cycle checkpoint * Changing gene expression so immune system can regconize cancer cells & destroy them 4. **Other New/Targeted Therapies** * Target oncogenes (kinases) * Mutated kinases can cause cancer * Kinase inhibitors * Oncolytic virus

Why are Transgenic Animals used?

* Test concepts in animals to **discover therapeutics** * **Test therapeutics** before human trials

1. cytogenetics/karyotyping 2. RFLP 3. Restriction enzymes 4. Southern Blotting

Methods to analyze DNA?\*

1. Northern Blotting 2. RPA

Methods to analyze RNA?\*

1. Western Blotting 2. IP & co-IP 3. ELISA

Methods to anaylze Proteins?\*