MCP 2 Flashcards
what is FISH and what is it used for?
fluorescent in situ hybridization
- DNA fragments with fluorescent labels are hybridized to chromosomes
- perform in metaphase or interphase cell
- either locus specific (single copy) or chromosome specific
- use control probe; 2 signals/chromosome in a normal person
what are the different types of FISH?
- repeat sequences: probes isolated from telomere or centromere region
- single copy: unique sequence; subtelomere is a specific type (high frequency of mutations there)
- chromosome painting: cocktail of probes, ID nonreciprocal translocation
restriction enzymes
based on ecoRI’s system of restriction and modification; recognition sequences can serve as landmarks in the DNA creating a DNA fingerprint; leaves sticky ends which can be ligated
gel electrophoresis
detect presence, size, and quantity of purified DNA; use polyacrylamide (smaller molecules and proteins) or agarose (larger molecules)
-anode attracts anions
DNA cloning steps
- restriction enzymes cut DNA fragments
- isolate with gel
- ligate into vector using T4 DNA ligase (bacteriophage enzyme)
- include amp so you can culture and see if DNA has ligated
types of cloning vector
- plasmids: 15 kb, circular DNA
- bacteriophage lambda: 20 kb, easy to purify because of viral size
- cosmid: 45kb, gutted lambda
- BAC: 100-300 kb, ds circular DNA, trick bacteria into thinking its their chromosome; low copy number; most applicable
- YAC: 100-2,000 kb, ds linear DNA; include telomere, centromere, and selectable marker; highly predisposed to recombination
cDNA library
contains only portions of the genome that becomes mRNA (introns removed)
-make RNA then degrade, reverse transcriptase to make DNA, DNA polymerase to make second DNA strand,
what is PCR
repeated rounds of mRNA primer directed DNA replication off a rare template used to amplify and detect specific sequences from a complex mixture of DNA
- heat to separate strands
- hybridize primers
- DNA synthesis from primers
- gel to see if it worked
multiplex PCR
amplify multiple specific sequences from a complex mixture in a single PCR reaction; used diagnostically (DMD)
reverse transcriptase PCR
make DNA copy of RNA genome then do PCR; so sensitive it can be used to detect presence of pathogen genes from patient samples (ex. HIV test)
southern blot
detects presence and size of a specific DNA sequence in a complex mixture; gel, transfer DNA to nitrocellulose using strong base, probe with hybridization for fragments with homolog to a sequence of interest
northern blot
purified mRNA is separated on a gel to see whether mRNA is expressed, what is the expression level, and are the mRNA of proper length
What is a microarray? What are some different types and what are they used for?
- compare two sets of DNA (normal and test, usually cDNA)
- miniaturized nucleic acid hybridization/detection
- isolate sequence, label, hybridize to DNA and read signal on microscope; each spot on gene chip is a probe that IDs a particular mutation
- first tier study in cases of unexplained developmental delay, intellectual disability, autism, and multiple congenital anomalies
- gene arrays: look for specific mutations or copy number variation
- chromosome arrays: used in clinical labs, look at locations and regions on chromosomes (peaks: duplications, valleys: deletion)
- expression arrays: look at mRNA to see heat maps of up regulation; characterize tissue types by the genes expressed; display by cluster analysis (genes that are coordinately regulated fall out into clusters)
denaturing vs. non denaturing gel electrophoresis
- denaturing: separates proteins by size, boil with SDS and ßmercaptoethanol (reducing agent) to create uniform (-), separate in polyacrylamide
- nondenaturing: use to compare change in charge or size between two proteins (ex. sickle cell)
isoelectric focusing
separate proteins by pI (proteins migrate until they carry no net charge)
western blot
detect presence, size, and abundance of a specific protein in a complex sample
-nitrocellulose incubated with primary antibody then washed and incubated with secondary antibody that has been coupled to an enzyme that fluoresces with substrate
primary antibody
antibodies raised in animals by injecting them with the antigen of interest
secondary antibody
antibodies raised against the constant regions of antibodies from different animal species; recognizes the primary antibody as foreign
monoclonal antibody
recognizes a specific epitope on a specific protein
immunofluorescence
detects presence and localization of a protein in a fixed tissue/cell sample
-use for analysis of dystrophin in muscle biopsies from normal vs. patients with DMD or beakers
GFP fluorescence
determine the localization and dynamics of a protein of interest after it has been fused to GFP; can look at living cells in real time
RNAi
introducing of dsRNA corresponding to a cellular mRNA induces degradation of mRNA and down regulation of the gene
- recognized by protein complex with RNA endonuclease “dicer” that cuts dsRNA into small pieces, RNA-protein complex destroys many RNA molecules
- morpholinos: ssDNA complementary to a transcript you want to knock down inhibits expression by blocking process of ribosome translation or by blocking splice site
fragile x (FRAXA)
disease from dynamic mutation, most common inherited mental retardation
- consider x-linked dominant with reduced penetrance
- CGG expansion in first exon and methylation of FMR1 gene
- anticipation (sherman paradox); means higher incidence in sons than uncles
- premutation females have mild symptoms, premature ovarian failure
- NTM: mild symptoms, 1/3 develop FX tremor/ataxia later
- NTMs may have FRAXA grandkids with full mutation but daughters will only have premutations
how do you test for fragile x?
southern blot
- repeats greater than 200 triplets become unstable; smear on southern blot
- premutation females: 4 bands because of x-inactivation
- full mutation male: only smear
sickle cell
structural varient hemoglobinopathy
- destruction of MnI site through mutation; do digest to distinguish S and C from A
- (HbA/HbS) asymptomatic carriers
- (HbS/HbS) sickle cell disease
thalassemia
imbalance of synthesis hemoglobanopathy, deficiency of one hemoglobin chain leads to inclusion bodies formed by excessive amount of the other chain
ßthalassemia
- reduced ß production; excess of aglobin chains form Heinz bodies in the RBCs, leads to hemolysis
- degres of allelic heterogeneity
- ß0 (severe) ß+ (reduced HbA detected)
athalassemia
- underproduction of aglobin chains
- homozygous deletion: hydrops fatalis
Hb Constant Spring
mutation results in tetramers of ßglobin chains which do not release O2 in peripheral tissues; aglobin is there but they don’t form tetramers
cystic fibrosis
autosomal recessive with allelic heterogeneity
- CFTR gene product is chloride channel across cell membranes in exocrine cells of lungs, pancreas, and sweat glands
- ∆F508 mutation is common
- therapy depends on class of mutation
fertilization
1 wk; in ampullary region of uterine tube, activates cleavage division; sperm and oocyte fuse and replicate (N–>2N; 23 mom, 23 dad)
cleavage division
mitotic divisions, increase number of cells, not total size
- cells are called blastomeres
- compaction occurs at 8 cell stage (segregates inner from outer cells, tight junctions stabilize
- 16 cell morula (totipotent)
blastocyst
occurs after morula undergoes cavitation; inner=ICM (pluripotent) and outer=trophoblast (placenta)
implantation
end of wk1; blastocyst expands in zone pellucida (zp prevents adhesion to oviduct), hatches in uterus and adheres to wall/begins implantation
- trophoblast differentiates into cyto (inner layer of mononucleated cells) and synctio (multinucleated layer that lacks distinct cell boundaries)
- ICM differentiates into epiblast and hypoblast to form bilaminar disc
gastrulation
- extensive cell movements and rearrangements of bilaminar disc form 3 germ layers
- node is organizer
- epiblast cells invaginate through primitive streak to form endo and meso
- body axes are establish before or during this time
neurulation
- neural crest–>delaminate and migrates to new locations, gives rise to many cell types; epithelial to mesenchymal transition
- neural plate–>neural tube–>brain and spinal cord; close cranial end (day 25) and posterior end (day 28)
- surface neural ectoderm–>skin (3 different ectoderm domains)
how do the neural crest cells give rise to characteristic cell types?
- 4 different regions of neural crest
- specific gene networks determine crest cell fate
- ex. cardiac neural crest cells enter the outflow tract of the heart to generate the septum between the great arteries
contiguous gene syndromes
regions in genome with clusters of closely associated genes whose normal functions are generally unrelated
william’s syndrome
- type of contiguous gene syndrome
- initially diagnosed as deletion of elastin gene on chromosome 7
- gene adjacent to elastin associated with learning and personality traits
velocardiofacial syndrome
- type of contiguous gene syndrome
- cleft palate and conotruncal heart defects
- chromosomes mispair because repeated sequences that flank the gene are similar (get micro duplication or deletion of 22q)
- variable phenotype
- wouldn’t be able to see this on karyotype
genome sequencing
ultimately want a genome-wide scan that will detect mutations with an individual’s medical concern; panels have been created that include all current data about particular genes or critical regions known to be associated with a specific disease
homologous genes
evolutionarily conserved; function interchangeably during development in different species
genome equivalence
all cells contain the same set of genes
differential gene expression
only a small percentage of the genome is expressed in each cell type; regulated at several levels and controls fundamental cellular processes
- proliferation
- specialization
- interactions
- movement
induction
one group of cells changes the behavior of an adjacent set of cells
- requires inducer and responders (must be competent)
- signals often transmitted between the two via paracrine (proteins secreted into ECM) or juxtacrine signaling
morphogens
paracrine signaling molecule that cause concentration-dependent effects; can specify more than one cell type by forming a concentration gradient
signaling cascades
many inductive molecules and morphogens transmit their signals through the cell membrane and to the cell nucleus via signal transduction pathways
- at every point in the cascade, receptors have to be competent
- transcription factor in nucleus binds DNA and alters gene expression
how is the left-right body axis formed?
-asymmetric signal cascade: cilia-driven fluid flow to left side triggers Nodal gene expression on the left side and establishes a morphogen gradient and signaling cascade in lateral plate mesoderm
–>activates downstream target genes (PITX2, Nodal, and nodal inhibitor Lefty)
odal gene codes for a member of TGFb superfamily, first expressed asymmetrically in the node region
-cilia generate fluid flow that
kartagener’s syndrome
immobility in what is meant to be motile cilia; accomplanied by a 50/50 chance of situs inversus or heterotaxy
mitochondrial diseases
- caused by mutations in oxidative phosphorylation, most series in CNS and muscle (neuropathies, encephalopathies, myopathies)
- may show autosome, x-linked or matrilineal inheritance
- must be a high proportion of mutants present to express dysfunction
- often progress with late onset
homoplasmy
populations o mitochondria that all have the same genetic composition; analygous to homozygosity in the nucleaus
heteroplasmy
when there are 2+ populations o fmitochondria present in a cell; analgoes to heterozygosity in the nucleus
replicative segregation
as cells divid, relative proportions of mutant mitochondria may change over time
acquired mutation
high mutation rate in mitochondrial DNA so it is possible for a mutation to occur as a new event and then proliferate in the population generating a subpopulation of mutants
