• Shuffle
    Toggle On
    Toggle Off
  • Alphabetize
    Toggle On
    Toggle Off
  • Front First
    Toggle On
    Toggle Off
  • Both Sides
    Toggle On
    Toggle Off
  • Read
    Toggle On
    Toggle Off
Reading...
Front

Card Range To Study

through

image

Play button

image

Play button

image

Progress

1/63

Click to flip

Use LEFT and RIGHT arrow keys to navigate between flashcards;

Use UP and DOWN arrow keys to flip the card;

H to show hint;

A reads text to speech;

63 Cards in this Set

  • Front
  • Back
What are the 3 basic elements of a plasmid vector?
1. origin of replication;

2. selectable marker gene (often antibiotic resistant genes);

3. lacZ gene with internal MCS for selection of plasmids that have the piece of DNA of interest. (inserted DNA disrupts LacZ gene)
X-Gal produces ____ color if the LacZ gene is intact in the plasmid.
blue
How does DNA sequencing work?
ddNTPs stop DNA polymerases. 4 mixes made for C,G,A or T reactions. Fragments are electrophoresed. The sequence is read from the bottom up of the gel and remember that the template strand will have the complementary sequence.
ddNTPs lack an OH group on what carbon?
3'
Sequencing using the same oligo primer is only good for about how many base pairs?
500 b.p.
What is site-directed mutagenesis?
Making mutations in the amino acid sequence to try to increase the ability of a protein.
Is PCR DNA methylated or unmethylated?
unmethylated.


Unmethylated DNA not cut when selecting for mutated plasmid.
Nettie Stevens Edmund Wilson...
Independently described the behavior of sex chromosomes-XX determines female; XY determines male.
Joe Hin Tjio determined that...
the number of chromosomes in humans is 46. In 1955
Jerome Lejune described...
that Down syndrome is caused by trisomy 21
The first genetic test for a disease was for what disease?
Huntington's
Newborn Screening
State mandated to detect cases ASAP -
Disorders where there is treatment available
Identify affected infants PRIOR to onset of irreversible findings
Must be a test that can distinguish affected infants from unaffected that
Can be done large scale – population screening
Inexpensive, reproducible, simple, “quick”

Examples: PKU, cystic fibrosis, sickle cell disease.
Carrier Screening
Recessive, monogenic disorders
cystic fibrosis
hemophilia


Selected (at-risk) population
Tay-Sachs disease
Sickle cell disease
Hemoglobinopathies
Appropriate screening for patients at increased risk to be carriers for hemoglobinopathies includes BOTH:

- Quantitative hemoglobin electrophoresis

- CBC with red cell indices
When is Prenatal Testing used?
To assess health of fetus, often when pregnancy is at increased risk of a genetic condition:

maternal age (>35)

family history

ethnicity

abnormal multiple marker (Quad) screen
Blood test during pregnancy to identify pregnancies at increased risk for certain birth defects

fetal ultrasound
When is Diagnostic Genetic Testing used?
Used to confirm or rule out a known or suspected genetic disorder in a symptomatic individual.
Bifid Uvula is Associated with a number of genetic conditions including:
22q deletion syndrome, familial cleft lip/palate, Smith-Lemli-Optiz syndrome
Williams syndrome is caused by a Microdeletion in an Elastin gene
of Chromosome 7p. It results in:
Cocktail party personality

Moderate mental retardation

Supravulvar Aortic Stenosis

Hypercalcemia

Dental anomalies: Short, square teeth and may be sparsely arranged.

Kidney anomalies
Osteogenesis Imperfecta causes:
Multiple fractures and brittle bone

Dentinogenesis imperfecta (DI): Brittle teeth.
Van der Woude is...
Most common cleft lip/ cleft palate syndrome

Autosomal dominant

* Bilateral lip pits
Gorlin syndrome results in
Jaw cysts and predisposition to cancers.
Predictive Testing is Offered to...
asymptomatic individuals with increased risk of a genetic disorder (i.e. with a family history of the disorder)
An Example of a High Utility Predictive Genetic Test is...
MEN2 – Multiple Endocrine Neoplasia, type 2


RET oncogene
Characterized by medullary thyroid carcinomas, pheochromocytomas
Childhood onset
Prophylactic thyroidectomy is SOC


Preventive intervention and clinically valid test
What is Genomics?
The study of genomes and includes sequencing DNA and collecting genomic variations within a population as well as transcriptional control of genes
What is Structural Genomics?
Genome sequencing, annotation, structural organization
What is Functional Genomics?
Transcriptomics and mutant analysis (some include functional proteomics)
What is Comparative Genomics?
Comparison and studies on the evolution of genomes
What are the limitations to DNA Microarrays?
Expense - Genomic Arrays $100,000 - $500,000

False Positives

Secondary Confirmation

Upregulated expression = upregulated production????

TOOOOO much information
What is Proteomics?
The scientific discipline of characterizing and analyzing the proteins, protein interactions, and protein modifications of an organism
What is Structural proteomics?
refers to the development and application of experimental approaches to define the primary, secondary, and tertiary structure of proteins (e.g. Edmund Deg, X-ray Crystallography, Molecular Fit Programs, etc.)
What is Functional proteomics?
refers to the development and application of global (proteome-wide or system-wide) experimental approaches to assess protein function by making use of the information provided by structural genomics
What is a 1D SDS-PAGE?
Fast, simple and reproducible tool for obtaining preliminary information on proteins

Proteins denatured and “coated” with SDS (-ve charge) then pushed through a polyacrylamide gel via electric current

MW proportional to 1/Speed of mobility
Mass Spectrometry
Two Components:

1. Ionization and Separation:
Matrix assisted laser desorption ionization (MALDI)
Electrospray ionization (ESI)

2. Mass Spectrometry:
MALDI – combined with Time of Flight (ToF)
ESI – combined with quadrupole MS


Derivation of Peptide Sequence
MALDI-ToF MS
Matrix – α-cyano-4-hydroxycinnamic acid or DHB

Laser desorption – N2 lasers 337nm

Ionization via photons (a little rough so small molecules to peptides)

Time of Flight:
Ions collected in a vacuum between two plates charged at 20,000 V
Cut power to one plate allows ions to travel down tube to a detector and the time of flight is recorded
ToF is proportional to m/z or MW
What are the benefits of Mass Spectrometry?
Automation

Low sample volume (nL)

Rapid (minutes)

Sensitive (nano-femtomoles)

Quantitation possible (area under the peak)

Multisamples (384)
What is Metabolomics?
The "systematic study of the unique chemical fingerprints that specific cellular processes leave behind" - specifically, the study of their small-molecule metabolite profiles
What is a Metabolome?
the collection of all metabolites in a biological organism, which are the end products of its gene expression
Metabolic profiling can give an instantaneous _______ of the physiology of that cell.
'snapshot'
What percentage of the US population is diagnosed with cancer during their lifetime?
25%
How many new cancer patients are diagnosed each year?
1.3 million
~75% of these patients receive systemic chemotherapy, however it will only provide a cure or long term remission in how many of these cases?
10%
What is the overall 5 year survival rate for cancer?
65%
As a cause of mortality, cancer is second only to...
cardiovascular disease
What are the criteria that distinguish transformed cells from normal cells in culture?
1. the requirement for serum in the cell culture medium to stimulate growth. Transformed cells have a reduced requirement for serum, approximately 10% that required for normal cells to grow.

2. the ability to grow without attachment to a supporting matrix (anchorage dependence). Normal cells (such as fibroblasts, smooth muscle cells) require adherence to a substratum (in this case, the bottom of the plastic dish) and will not grow if suspended in a soft agar mixture (the consistency) of loose jello. Transformed cells, however, have lost this anchorage dependence for growth
The test used to screen a chemical for potential carcinogenicity is called...
Ames assay
How does the Ames assay work?
(1) Basis for test: Salmonella typhimurium with mutations (His-) unable to code for one enzyme in histidine synthetic pathway, thus needs histidine in medium (2) Procedure: test chemical for potential to cause mutation at His- site changing it to His+ allowing bacteria to grow without histidine in medium

(3) Problem: bacteria not contain spectrum of monooxygenases found in higher animals, thus compound requiring activation to become mutagenic not occur in bacteria (a) Solution: incubate agents to be tested with postmitochondrial supernatant of rat liver (S-9 fraction) which contains various enzymes to activate potential mutagens & carcinogens
The genes involved in the development of cancer are classified as...
oncogenes or tumor suppressor genes
mutated derivatives of normal genes whose function is to promote proliferation or cell survival
Oncogenes
proto-oncogenes can code for:
A. Growth factors
B. Growth factor receptors
C. Signal transduction proteins
D. Intracellular kinases
E. Transcription factors
Mutant p53 is present in what percent of cancers?
75%
P53 is located on the small arm of ________ and is present in all normal tissues
Chromosome 17
the most common genetic change in human cancer is what?
Mutation of p53
When a mutant allele inactivates the normal one, it is called a...
dominant negative gene
p53 is an important tumor suppressor and it works what 3 ways?
Preventing division of cells:
Arrests cells in G1 stage to allow time for DNA repair

Triggers apoptosis: If irreparable, triggers apoptosis

Production of regulatory molecules: helps slice specific RNA into small regulatory molecules called microRNAs
Most mutations occur in the ________ domain of p53
DNA-binding
Does p53 play a role in primary RNA transcript production?
No.


p53 mutations disrupt the machinery that chops RNA into microRNA
p53’s DNA-binding domain interacts with what other two proteins?
Drosha and p68 are proteins in an assemblage responsible for snipping primary transcripts into hairpin-shaped molecules
True/False?
Mutant p53 is worse than no p53
True


Normal p53 serves to brings Drosha and p68 together
Altered p53 keeps the two apart and interrupts their normal interaction
True/False?
A cell population must accumulate multiple mutations for transformation to malignant cells.
True
Apoptosis is initiated by either
death receptor activation

or

intracellular signals leading to release of the mitochondrial protein, cytochrome c.
Around ____ of the deaths in the United States each year are caused by cancer
20%
tumor-generating cells share key traits with _____ cells, including an unlimited life span and the ability to generate a diverse range of other cell types
stem
Acute myeloid leukemia (1994)
Acute lymphoblastic leukemia (1997)
Chronic myelogenous leukemia (1999)
Breast (2003)
Multiple myeloma (2003)
Brain (2004)
Prostate (2005)
malignant stem cells have been identified to date