Exam #5: Molecular Medicine I Flashcards Preview

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Flashcards in Exam #5: Molecular Medicine I Deck (47):

Direct Sequencing (Sample type & what it detects)

- DNA sample
- Detects point mutations/SNPs


Whole Exome Sequencing (Sample type & what it detects)

- DNA sample
- Detects point mutations/ SNPs in exons- not introns or intergenic regions


SNP Typing (Sample type & what it detects)

- DNA sample
- Detects SNPs in the entire genome


PCR (Sample type & what it detects)

- DNA sample
- Detects insertions or deletions


Reverse Transcriptase PCR(Sample type & what it detects)

- RNA sample
- Detects the expression levels of a small number of genes


Allele Specific Oligonucleotides (Sample type & what it detects)

- DNA sample
- Detects point mutations & small insertions/deletions


Gene Expression Array (Sample type & what it detects)

- RNA sample
- Detects expression levels of thousands of genes- not intergenic regions or introns


Methylated DNA PCR (Sample type & what it detects)

- DNA sample
- Detects epigenetic changes/ DNA methylation


Comparative Genome Hybridization (Sample type & what it detects)

- DNA sample
- Detects insertions, deletions, & aneuploidies in the kilo-to megabase range


FISH(Sample type & what it detects)

- DNA sample
- Detects copy number of a selected chromosomal region


ELISA(Sample type & what it detects)

- Protein sample
- Detects the amount of protein in a sample


Western Blot (Sample type & what it detects)

- Protein sample
- Detects the amount & size of protein in a sample


Direct Sequencing

Sequencing the genome for an exact mutation


Indirect Marker Analysis

When the error in the gene responsible for causing a disease has not been identified, "markers" are used to find out whether a person has inherited the region of the genetic code that is passing through the family with the disease. Markers are DNA sequences located close to the area of interest that are so close that they are almost always inherited together with the disease. When markers are this close to a gene where they are inherited together, they are said to be "linked." In this way, if someone has the set of linked markers, he or she will also have the disease-causing gene. For this reason, these types of studies are also called "linkage studies."



- Whole genome sequencing
- Not feasible or useful in the clinical setting
- Analyzing 3 billion base pairs



- Whole exome sequencing
- Only 3% of the entire genome codes for exon sequences
- Sequences the exons only


SNP typing

- 99% of the DNA does not vary between individuals
- To learn about the genetic characteristics of an individual, look at the single nucleotide polymorphisms only (the different stuff)


What is the importance of copy number?

- Individuals don't just vary by single nucleotide polymorphisms, but also by small insertions/deletions of the chromosome (i.e. duplication of a gene on a chromosome)
- This phenomenon is referred to as copy number variation


What are the two techniques used to detect copy number?

1) Comparative Genome Hybridization
2) Flourescence in situ Hybridization= allows identification of a chromosomal locus by using a specific fluorescent probe


What are the limits of DNA sequencing?

- Will not show what chromosome the mutation is on
- Will not show a deletion or insertion


Explain PCR & its application in the analyses of polymorphisms & gene expression.

Polymerase Chain Reaction

- Used to amplify DNA fragments
- Mutations can be detected by using wild-type or mutant primers
- Insertions or deletions can be detected because they change the length of the amplified fragment



- Allele Specific Oligonucleotides
- Detect single nucleotide polymorphisms & mutations
- Oligonucleotide primer paired with mutant and wild-type DNA


What is the difference between ASO arrays & gene expression arrays?

- ASO array= Oligonucleotide primers (mutant) printed on a plate & DNA hybridized-- allows for genome screening for common mutations
- Gene Expression Array= Oligonucleotide primers bind mRNA transcripts, NOT DNA
- Indicates expression level of a gene


What are the potential applications of pharmacogenomics?

- Field that correlates genotypes & individual drug responses
- Polymorphisms can be present in genes coding for drug targets
- Polymorphisms can be in the enzymes that metabolize drugs


What is prenatal diagnosis & what two techniques are used in prenatal diagnosis?

Diagnosis of a genetic condition before birth

- Amniocentesis
- Chorionic villus sampling


What is direct to consumer genetic testing?

Genetic testing marketed directly to consumers


What are the ethical problems with direct to consumer genetic testing?

Results of genetic screening should never be presented without adequate counseling


How are polyclonal antibodies produced?

1) Purify protein of interest
2) Inject into rabbit
3) Withdraw blood
4) Rabbit serum contains multiple different antibodies to the protein (to different epitopes of the antigen)


How are monoclonal antibodies produced?

1) Same initial steps as polyclonal antibodies
2) Remove spleen cells
3) Fuse spleen cells with myeloma cells grown in culture (each will grow a specific antibody for an epitope)
4) Select & grow hybrid cells making desired antibody


How are antibodies used in ELISAs?

- Sandwich ELISA= detection of an antigen
- Indirect ELISA= detection of antibody


How are antibodies used in western blotting?

Technique that uses specific antibodies to indicate the molecular weight of a protein

-Tells if the protein is present or not
- Used to quantify the amount present
- Shift in banding can identify a modification of the protein in question


How are antibodies used to treat cancer?

- Herceptin was the first monoclonal antibody approved for treatment of metastatic breast cancer
-inhibits ErbB2/ HER2/Neu receptor tyrosine kinase located on cell surface


What will DNA sequencing show & not show?

- Will show if the person is homozygous or heterozygous
- Will NOT show which chromosome a mutation is on
- Will NOT show a deletion or duplication


Comparative Genome Hybridization (CGH)

1) Obtain ss-DNA from patient & control
2) Label both with different dye
3) Mix & hybridize to metaphase chromosomes
4) Uneven labeling (color) indicates an insertion or deletion (variation in copy number)


What are the steps in PCR?

1) Denature DNA (want ss-DNA)
2) Synthesize oligonucleotides that are complimentary to the ends of the DNA fragment to be studied (primers)
3) Primers anneal to ss-DNA
4) Polymerase synthesizes complimentary strand
5) Repeat


Real-time PCR

Allows for quantification of amounts of nucleic acids

- Measures amplified DNA after each cycle
- Determined how many cycles are required to pass a threshold
-Template amount can be quantified by the number of cycles required to reach threshold
- More cycles= less template & vice versa


Reverse-Transcriptase PCR

Used for RNA analysis

- Reverse transcription of RNA to DNA by reverse transcriptase
- Makes cDNA
- cDNA serves as the template for the PCR reaction


Warfarin example of pharmacogenetics

- Oral anticoagulant that inhibits vitamin K epoxide reductase
- polymorphism in p450 (CYP2C9) causes slow metabolism & higher sensitivity
- polymorphism in vitamin K epoxide reductase (VKORC1) that leads to warfarin tolerance


What cells produce antibodies?



What is a sandwich ELISA routinely used for?

The detection of antigen or peptide in human serum

1) Parathyroid Hormone
2) HbA1c
3) CRP


What is an indirect ELISA routinely used for?

Diagnosis of autoimmune disease

- RA
- Also, HIV


What are the steps in a western blot?

1) Separate protein extracts by electrophoresis
2) Transfer proteins to membrane
3) Add enzyme-linked specific antibody
4) Antibodies bind antigen (proteins)
5) Detect label in band pattern


Gene Therapy

Insertion of a functional copy of a gene into mutant cells


What is the biggest obstacle to gene therapy?



What viruses are used as vectors in gene therapy?

1) Retrovirus
2) Adenovirus
3) Adeno-associated virus (No known disease)
4) HSV-1


What are the conflicting properties of a viral vector?

- Virus should spread aggressively through the body; however, if TOO AGGRESSIVE, can trigger sepsis
- Integration into the host genome, which can lead to cancer (leukemia in clinical trials)


What are the four non-viral vectors in gene therapy?

1) Liposomes= lipid vesicle w/ DNA payload
2) Naked DNA
3) Complexed DNA
4) Artificial Chromosomes