Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

Week 7 > Molecular Biology in Medicine > Flashcards

Molecular Biology in Medicine Flashcards

1
Q

What are the 4 Methods to analyze DNA?*

A
  1. cytogenetics/karyotyping
  2. RFLP
  3. Restriction enzymes
  4. Southern Blotting
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are the 2 Methods to analyze RNA?*

A
  1. Northern Blotting
  2. RPA
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the 3 Methods to anaylze Proteins?*

A
  1. Western Blotting
  2. IP & co-IP
  3. ELISA
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

This is an example of what molecular biology method?

A

Cytogenetics/Karyotyping

  • Early method of genetic testing
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How can we identify single nucleotide pair mutations?

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

How do restriction enzymes/digestion function?

A
  • 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’

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are the 6 steps in a Southern Blot?

A
  1. Detect specific DNA sequence
  2. Cut DNA w/ restriction enzymes
  3. Run agrose gel (electrophoresis)
    • separate large charged molecules based on size
    • optional: acid treat gel to break up large fragments/enhance transfer
  4. Transfer to filter
  5. Hybridize labeled probes
  6. Detect on film/machine
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is hybridization?

A
  • 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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What are the 4 steps of a Northern Blot?

A
  1. Run RNA on denaturing agrose gel
  2. Transfer RNA to membrane
  3. Hybridize to labeled probe
  4. 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the Ribonuclease Protection Assay (RPA) procedure?

A
  1. Hybridize target RNA w/ labeled probes
  2. Digest non-hybridized RNA w/ RNAase
  3. Run on denaturing polyacrylamide gel
  4. 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Why use a Western Blot?

A
  • 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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Why use a Dot Blot?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Other detection methods:

In situ hybridization?

A
  • Detect DNA or RNA sequences in tissue or cells
    • Use of labeled nucleic acid probes

Note: Useful in pathology

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Other detection methods:

Immunohistochemistry?

A
  • Detect proteins in tissue
    • via use of antibodies

Note: Useful in pathology

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Other detection methods:

Immunocytochemistry?

A
  • Detect proteins in intact cells (usually with most or all of the extracellular matrix removed)
    • via use of antibodies

Note: Useful in pathology

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is Enzyme-Linked Immunosorbent Assay (ELISA)?

A
  • Detects proteins, pathogens, etc
    • qual or quantitative
    • quick & easy

Sandwich ELISA:

  1. Plate coated w/ capture Ab
  2. Sample added
  3. Detecting Ab added, binds to Ag
  4. Enzyme-linked 2° Ab added & binds to detecting Ab
  5. Substrate added, converted by enzyme to detectable form
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Why use Chromatin Immunoprecipitation (ChIP)?

A
  • 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
18
Q

Why use a Polymerase Chain Reaction (PCR)?

A
  • Requires:
    • template DNA
    • primers
    • polymerase
    • nucleotides
  • Detects & amplify specific DNA
    • product doubles w/ each cycle

Process:

  1. Denaturation (~94°C) - 5 min
  2. Annealing (~55°C) - 20-40 cycles
  3. 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

19
Q

Why use Reverse Transcription PCR (RT-PCR)?

A
  • 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)
20
Q

Why use Real Time PCR?

A
  • 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:

  1. In intact probes, reporter fluorescence is quenched
  2. Probes & complementary DNA strand = hybridized and reporter fluorescence is still quenched
  3. During PCR, the probe is degraded by the Taq polymerase
    • fluorescent reporter released
21
Q

Why use a Microarray?

A
  • 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
22
Q

What are the steps for DNA Cloning?

A
  1. Make cDNA from (fully mature) mRNA
    • Use reverse transcriptase
  2. Digest cDNA & vector (plasmid)
    • expression vector
  3. Ligate cDNA fragment into vector
    • intro-dependence problem
    • need natural intronless RNA help

Genomic library

  • collection of total genomic DNA from a single organism
23
Q

What are Reporter Systems?

A
  1. Regulatory sequence to be studied
    1. ex: gene promoter
  2. Reporter gene
    1. encoding GFP OR Luciferase
  3. 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
24
Q

Why use a Plasmid/Expression Vector?

A
  • Can put cDNA into a plasmid/expression vector
    • will see the expression/effect of DNA
  • Allows for transfection
    • introduction of exogernous DNA into cells
25
Q

What are the 2 types of Transfection?

A
  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)
26
Q

What is Sanger Method?

A
  • 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
27
Q

What is Next Generation Sequencing?

A
  • 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
28
Q

What is a Single Nucleotide Polymorphisms?

A
  • Change of a nucleotide at a single base-pair on DNA
    • Other detections:
      • Indels (insertions/deletion of bp)
  • Also be detected by Microarrays
29
Q

Why use Circulating DNA Diagnostics?

A
  • 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
30
Q

Types of DNA biomarker detection?*

A
  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
31
Q

Strategies for Gene Therapy?

A
  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
32
Q

Types of Cancer Immunotherapy

A
  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
  1. Other New/Targeted Therapies
  • Target oncogenes (kinases)
    • Mutated kinases can cause cancer
  • Kinase inhibitors
  • Oncolytic virus
33
Q

Why are Transgenic Animals used?

A
  • Test concepts in animals to discover therapeutics
  • Test therapeutics before human trials
34
Q
  1. cytogenetics/karyotyping
  2. RFLP
  3. Restriction enzymes
  4. Southern Blotting
A

Methods to analyze DNA?*

35
Q
  1. Northern Blotting
  2. RPA
A

Methods to analyze RNA?*

36
Q
  1. Western Blotting
  2. IP & co-IP
  3. ELISA
A

Methods to anaylze Proteins?*

37
Q
  • 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
A

What is Sanger Method?

38
Q
  • 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
A

What is Next Generation Sequencing?

39
Q
  • 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
A

Why use Circulating DNA Diagnostics?

40
Q
  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
A

Types of DNA biomarker detection?*

41
Q
  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
A

Strategies for Gene Therapy?

42
Q
  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
  1. Other New/Targeted Therapies
  • Target oncogenes (kinases)
    • Mutated kinases can cause cancer
  • Kinase inhibitors
  • Oncolytic virus
A

Types of Cancer Immunotherapy

Week 7 (10 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions