Flashcards in Biochem Lab Techniques Deck (51):
Steps in PCR
1. Denaturation (heating)
2. Annealing (DNA primers anneal to specific sequence on each strand to be amplified)
3. Elongation (heat stable DNA polymerase replicates DNA sequence following each primer)
What is PCR useful for?
Used to amplify a specific fragment of DNA; useful as diagnostic tool
DNA probe, DNA sample
DNA probe, RNA sample
What is Northern blot useful for?
Studying mRNA levels (which are reflective of gene expression)
Antibody probe, protein sample
Oligonucleotide probe, DNA binding protein sample
What are microarrays used for?
Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments
Able to detects SNPs and copy number variations (CNVs)
Used to detect a specific antigen or antibody in a patient's blood sample
Difference between direct and indirect ELISA
Direct - use known antibody to identify presence of antigen in patient's blood; Antibody is directly coupled to color generating enzyme
Indirect - uses test Ag/Ab to detect specific Ag/Ab and then secondary antibody coupled to color generating enzyme is added to detect Ag/Ab complex
What is FISH?
Fluorescent DNA/RNA probe binds to specific gene interest on chromosomes --> used for specific localization of genes and direct visualization of anomalies like micro deletions at molecular level
Steps of cloning (5)
1. Isolate eukaryotic mRNA of interest
2. Expose mRNA to reverse transcriptase to produce cDNA
3. Insert cDNA fragments into bacterial plasmids containing Ab resistance genes
4. Transform recombinant plasmid into bacteria
5. Surviving bacteria on antibiotic medium produce cloned DNA
How is cDNA different than DNA?
It doesn't contain introns
What is cre-lox system?
Can inducibly manipulate genes at specific developmental points to study a gene whose deletion causes embryonic death
What is RNA interference?
dsRNA is synthesized that is complementary to target mRNA --> when transfected into human cells the dsRNA separates and promotes degradation of target mRNA --> knocking down gene expression
What is codominance?
Both genes contribute to phenotype of heterozygote (blood groups, alpha 1 antitrypsin deficiency)
What is variable expressivity?
Phenotype varies among individuals with same genotype (2 patients with NF1 have varying severity)
What is incomplete penetrance?
Not all individuals with mutant genotype show the mutant phenotype (BRCA1 mutation)
What is Pleiotropy?
One gene contributes to multiple phenotypic effects (PKU causes light skin, intellectual disability, musty body odor)
What is Anticipation?
Increased severity of earlier onset in succeeding generations (Trinucleotide repeat diseases)
What is loss of heterozygosity?
It a patient inherits/develops a mutation in a tumor suppressor gene, the complementary allele must be deleted or mutated before cancer develops (two hit hypothesis)
Two hit hypothesis is an example of?
Loss of heterozygosity
What is dominant negative mutation?
Exerts a dominant effect; a heterozygote produces a non-functional altered protein that also prevents the normal gene product from functioning (mutation of transcription factor in its allosteric site that prevents wild type transcription factor from binding)
What is linkage disequilibrium?
Tendency for certain alleles at 2 linked loci to occur together more or less often than expected by chance (measured in population)
What is mosaicism?
Presence of genetically distinct cell lines in same individual
1. Somatic mosaicism = mutation arises from mitotic errors after fertilization and propagates through multiple tissues/organs
2. Gonadal mosaicism = mutation only in egg/sperm cells = mutation only in offspring
Example of mosaicism?
McCune Albright syndrome (lethal if mutation occurs before fertilization)
What is locus heterogeneity?
Mutations at different loci can produce a similar phenotype - Albinism
What is allelic heterogeneity?
Different mutations in same locus produce the same phenotype - B thalassemia
What is heteroplasmy?
Presence of both normal and mutated mtDNA; resulting in variable expression in mitochondrially inherited disease`
What is uniparental disomy?
Offspring receives 2 copies of chromosome from one parent and no copies from the other
What is uniparental heterodisomy?
Meiosis I error - offspring receives 2 copies of chromosome from one parent (same chromosomes as parent)
What is uniparental isodisomy?
Meiosis II error or post zygotic chromosomal duplication of one pair of chromosomes (2 copies are identical!)
Hyperphagia, obesity, intellectual disability, hypogonadism and hypotonia
Prader Willi syndrome (maternal imprinting; paternal gene is mutated/deleted OR maternal uniparental disomy)
Inappropriate laughter, seizures, ataxia, severe intellectual disability
Angelman syndrome (paternal imprinting, maternal gene is deleted/mutated)
Inheritance pattern of enzyme deficiencies (usually)
Inheritance pattern of defects in structural genes
Inheritance pattern of hypophosphatemic rickets
X linked dominant
Conditions that are polygenic
Ischemic heart disease
Type II DM
Mutation causing ADPKD
85% due to mutation in PKD1 on chromosome 16
Remainder from PKD2 on chromosome 4
Mutation causing Familial adenomatous polyposis
Mutation in chromosome 5q (APC gene)
Mutation in Familial hypercholesterolemia
Defective or absent LDL receptor --> elevated LDL
Mutation in Hereditary hemorrhagic telangiectasia
Also known as Osler-Weber-Rendu syndrome
Mutation in Hereditary Spherocytosis
Mutation in spectrin or ankyrin
Mutation in Huntington disease
on Chr 4; trinucleotide repeat CAG
Mutation in Li Fraumeni
Abnormal Tp53 (sarcoma, breast, leukemia, adrenal gland cancer)
Mutation in Marfan's
FBN1 mutation on chromosome 15 --> defective fibrillin
Mutation causing MEN2A and MEN2B
Mutation in RET gene
Mutation causing NF1 (von Recklinghausen disease)
Mutation in NF1 gene on chromosome 17
Mutation causing NF2
Mutation in NF2 gene on chr. 22
Mutation causing tuberous sclerosis