Final Week 2 Flashcards
What major restriction endonuclease is commonly used?
EcoRI
What is required for the ends of plasmids and DNA fragment to be “glued” together?
ATP and Ligase
What is agarose gel used for?
separates population of DNA or RNA molecules based on size
DNA is already charged
What is SDS-PAGE used for?
separate population of proteins based on size
Adds negative charge to every protein to open them up
What is used to pull out all mRNA from a cell to make cDNA?
Poly A tail with poly T-primer
What can PCR be used for?
amplifying genes
Used to detect altered genes or foreign DNA
What method is used in forensics?
Short tandem repeats PCR
CACCA or GTGTGT repeats
What change can be used to find mutations?
RFLPs
What can be used to determine variations in gene expression?
DNA microarray
More intense signal->higher degree of binding of probe->higher level of expression
What is the advantage of using QPCR?
analyzing PCR in real time. Analyze different genes in same sample.
Faster the fluorescence shows up the higher amounts of target DNA of cell sample being created.
Show how many copies are being made in a person vs only a pos/neg result
What is an epitope?
Specific site on antigenic molecule recognized by Fab
What does indirect Elisa detect?
Presence of Ab to specific antigen
HIV
What does sandwich elisa detect?
Detection and quantitation of an antigen
Troponin-1
Pregnancy - Hormone immunoassays
What does western blotting used for?
compare protein levels in samples
Monoclonal or polyclonal - level and size of products
what two enzymes are most commonly used with antibodies? What do the enzymes catalyze?
HRP and AP
HRP catalyzes the oxidation of substrates (DAB) by hydrogen peroxide and Brown precipitate is product
AP hydrolyzes phosphate group from substrate (BCIP/NBT) and gives off dark blue/purple product
What is proteomics used for? How?
Dont know what proteins are altered, look for unknown
Protein expression and/or alteration and post-translational modification
Isoelectric focusing used and then electrophoresis with 2D-DIGE. Scanned and then use Mass spectrometry to identify
How is mass spectrometry done?
Used to identify unknown proteins. Chops them up into pieces with tryptic digestion. Then ionization, detection method and computer data base with known protein sizes
What is competence?
capacity to respond to inductive signal
What is the structure of homeodomain proteins? What is the general function?
Helix-turn-helix and the homeobox is involved in the regulation of patterns of morphogenesis
What are paralogs?
Genes related by duplication within a genome, each with a different function
Describe Hox genes
Transcription factors
Role in craniocaudal segmentation of body
3’ to 5’ expression order
Lay out big pattern
Retinoic acid: acts through Hox genes and serves as intercellular signaling molecule that guides development of posterior portion of embryo
Mutations: loss- posterior->anterior
Gain - Anterior->posterior
What happens if there is too much retinoic acid?
Too much vitamin A causes posterior part of body to have issues - not form or major deformities
What are Pax genes?
TFs-homeodomain
Role in formation of tissues and organs; nervous system/sense organs, epithelial-mesenchyme transition
maintain normal function after birth
What is Aniridia?
Pax6 mutation
Complete or partial absence of iris
What are Lim proteins?
TFs-homeodomain
Some bind to DNA some to cytoplasm
Involved in formation of virtually all body segments
What happens if there is an absence of Lim Proteins?
Headless
What are Dlx genes?
TFs-homeodomain
Patterning of outgrowing appendages in early embryogenesis
Morphogenesis of jaw and inner ear
What are Msx genes
TFs - homeodomain
Prenatal- inhibit cell differentiation
Postnatal-maintain proliferative capacity of tissue
Face, limb epithelial-mesenchymal interaction in face and limbs
What are T-box genes
TFs
Mesodermal germ layer development and specifying whether limbs will be forelimb or hindlimb
What do basic helix-loop-helix genes regulate?
TFs that regulate myogenesis - muscle tissue
What are forkhead proteins?
TFs that regulate expression of genes involved in cell growth, proliferation, differentiation, and longevity. Pioneering transcription activity - bind condense chrromatin
What happens if there is a disorder in Foxhead gene?
Disorder in speech and language development
What is a zinc finger?
TFs with Cys and His linked to Zinc
Regulate myogenesis, insert into DNA
Bone, cartilage, and tooth development
What happens if there is a zinc deficiency (Zinc Finger)?
Skeletal growth retardation; osteoporosis
What happens if there is a mutation in the SOX?
Abnormal development of tissues, wide variety of signs and symptoms
SOXA = SRY
Reproductive developmental problems along with skeletal growth retardation
What is WTI?
Wilms’ tumor suppressor gene
Kidney and gonadal development
Defect-urogenital abnormal
What is TGF-beta? Give an example
Transforming growth factor
BMP - block the action of neural formation
Regulatory molecules: noggin, chordin, WNT, FGF
What are FGFs?
Fibroblast GFs-lots of variation
Angiogenesis, wound healing, embryonic development (limbs/brain) and various endocrine signaling pathways
Closely associated with ECM and bind to heparan sulfate to activate
What are the three types of hedgehogs? What is the receptor?
Desert, Indian, and sonic
Patched (PTCH)
What is PTCH bound to?
Smoothened transmembrane protein (SMO)
What is the function of SHH?
Regulating vertebrate organogenesis, facial and limb patterning and organization of brain
Remains important in adult by controlling cell division of stem cells