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Flashcards in Biochem Lab Techniques Deck (51):
1

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)

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What is PCR useful for?

Used to amplify a specific fragment of DNA; useful as diagnostic tool

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DNA probe, DNA sample

Southern blot

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DNA probe, RNA sample

Northern blot

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What is Northern blot useful for?

Studying mRNA levels (which are reflective of gene expression)

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Antibody probe, protein sample

Western blot

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Oligonucleotide probe, DNA binding protein sample

Southwestern blot

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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)

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Used to detect a specific antigen or antibody in a patient's blood sample

ELISA

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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

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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

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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

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How is cDNA different than DNA?

It doesn't contain introns

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What is cre-lox system?

Can inducibly manipulate genes at specific developmental points to study a gene whose deletion causes embryonic death

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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

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What is codominance?

Both genes contribute to phenotype of heterozygote (blood groups, alpha 1 antitrypsin deficiency)

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What is variable expressivity?

Phenotype varies among individuals with same genotype (2 patients with NF1 have varying severity)

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What is incomplete penetrance?

Not all individuals with mutant genotype show the mutant phenotype (BRCA1 mutation)

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What is Pleiotropy?

One gene contributes to multiple phenotypic effects (PKU causes light skin, intellectual disability, musty body odor)

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What is Anticipation?

Increased severity of earlier onset in succeeding generations (Trinucleotide repeat diseases)

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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)

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Two hit hypothesis is an example of?

Loss of heterozygosity

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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)

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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)

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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

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Example of mosaicism?

McCune Albright syndrome (lethal if mutation occurs before fertilization)

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What is locus heterogeneity?

Mutations at different loci can produce a similar phenotype - Albinism

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What is allelic heterogeneity?

Different mutations in same locus produce the same phenotype - B thalassemia

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What is heteroplasmy?

Presence of both normal and mutated mtDNA; resulting in variable expression in mitochondrially inherited disease`

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What is uniparental disomy?

Offspring receives 2 copies of chromosome from one parent and no copies from the other

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What is uniparental heterodisomy?

Meiosis I error - offspring receives 2 copies of chromosome from one parent (same chromosomes as parent)

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What is uniparental isodisomy?

Meiosis II error or post zygotic chromosomal duplication of one pair of chromosomes (2 copies are identical!)

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Hyperphagia, obesity, intellectual disability, hypogonadism and hypotonia

Prader Willi syndrome (maternal imprinting; paternal gene is mutated/deleted OR maternal uniparental disomy)

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Inappropriate laughter, seizures, ataxia, severe intellectual disability

Angelman syndrome (paternal imprinting, maternal gene is deleted/mutated)

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Inheritance pattern of enzyme deficiencies (usually)

Autosomal recessive

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Inheritance pattern of defects in structural genes

Autosomal dominant

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Inheritance pattern of hypophosphatemic rickets

X linked dominant

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Conditions that are polygenic

Androgenetic alopecia
Epilepsy
Ischemic heart disease
Schizophrenia
Type II DM
HTN
Glaucoma
Malignancy

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Mutation causing ADPKD

Autosomal dominant
85% due to mutation in PKD1 on chromosome 16
Remainder from PKD2 on chromosome 4

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Mutation causing Familial adenomatous polyposis

Autosomal dominant
Mutation in chromosome 5q (APC gene)

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Mutation in Familial hypercholesterolemia

Autosomal dominant
Defective or absent LDL receptor --> elevated LDL

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Mutation in Hereditary hemorrhagic telangiectasia

Also known as Osler-Weber-Rendu syndrome
Autosomal dominant

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Mutation in Hereditary Spherocytosis

Autosomal dominant
Mutation in spectrin or ankyrin

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Mutation in Huntington disease

Autosomal Dominant
on Chr 4; trinucleotide repeat CAG

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Mutation in Li Fraumeni

Autosomal Dominant
Abnormal Tp53 (sarcoma, breast, leukemia, adrenal gland cancer)

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Mutation in Marfan's

Autosomal dominant
FBN1 mutation on chromosome 15 --> defective fibrillin

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Mutation causing MEN2A and MEN2B

Autosomal dominant
Mutation in RET gene

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Mutation causing NF1 (von Recklinghausen disease)

Autosomal dominant
Mutation in NF1 gene on chromosome 17

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Mutation causing NF2

Autosomal dominant
Mutation in NF2 gene on chr. 22

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Mutation causing tuberous sclerosis

Autosomal dominant

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Mutation causing von Hippel Lindau disease

Autosomal dominant
deletion of VHL gene on chromosome 3p