Usmle Step 1: Biochemistry 4

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Polymerase Chain Reaction (PCR)

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- molecular biology lab procedure used to synthesize many copies of a desired fragment of DNA

- Steps:
1. DNA is denatured by heating to generate 2 separate strands
2. During cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified
3. Heat-stable DNA polymerase replicates the DNA sequence following each primer
These steps are repeated multiple times for DNA sequence amplification.

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Southern/Northern/Western blots and Microarrays

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- Southern blot: A DNA sample is electrophoresed on a gel and then transferred to a filter. The filter is then soaked in a denaturant and subsequently exposed to a labeled DNA probe that recognizes and anneals to its complementary strand. The resulting double-stranded labeled piece of DNA is visualized when the filter is exposed to film.

- Norther blot: Similar to Southern blot except it involves radioactive DNA probe binding to sample RNA

- Western Blot: Sample protein is seperated via gel electrophoresis and transfered to a filter. Labeled antibody is used to bind to relevant protein.

- Microarrays: Thousands of nucleic acid sequences are arranged in grids on glass or silicon. DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amounts of complementary binding.

- SNoW DRoP: Southern (DNA), Northern (RNA), Western (Protein)

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Enzyme-linked immunosorbent assay (ELISA)

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- A rapid immunologic technique testing for antigen-antibody reactivity

- Patient's blood sample is probed with either a test antigen or test antibody (both of which are physically coupled to a color generating enzyme). If the target substance (antibody or antigen of interest) the sample will undergo a color change indicating a positive result.

- Used to test for the presence of HIV antibody in patient's blood sample.

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Fluorescence in situ hybridization (FISH)

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- Fluorescent probe binds to specific gene site of interest
- Specific localization of genes and direct visualization of anomalies (e.g. microdeletions) at the molecular level.

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Sanger DNA Sequencing

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- Dideoxynucleotides halt DNA polymerization at each base, generating sequences of various lengths that encompass the entire sequence. The terminated fragments are electrophoresed and the original sequence can be deduced.

- The polymerization is carried out in four separate containers each containing only 1 of the four dideoxynucleotides (corresponding to either A,T,C,G).

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Codominance

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neither of 2 alleles is dominant (e.g., blood groups)

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Variable expression

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nature and severity of the phenotype varies from one individual to another

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Pleiotropy

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1 gene has more than 1 effect on an individual's phenotype

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Imprinting

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differences in phenotype depend on whether the mutation is of maternal or paternal origin (e.g., AngelMan’s syndrome [Maternal], Prader-Willi syndrome [Paternal])

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Anticipation

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Severity of disease worsens or age of onset of disease is earlier in succeeding generations (e.g., Huntington's disease)

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Loss of heterozygosity

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- If a patient inherits or develops a mutation in a tumor suppressor gene, the complementary allele must be deleted/mutated before cancer develops

- This is not true of oncogenes

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Incomplete penetrance

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Not all individuals with the mutant genotype show the mutant phenotype.

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Dominant negative mutation

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mutation has a dominant effect meaning the heterozygote produces a nonfunctional altered protein that also prevents the normal gene product from functioning (common in multisubunit gene products)

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Linkage disequilibrium

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- Tendency for certain alleles at 2 linked loci to occur together more often than expected by chance

- Measured in a population, not a family, and often varies in different populations.

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Mosaicism

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Occurs when cells in the body have different genetic makeup (e.g., lyonization - random X inactivation in females)

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Locus heterogeneity

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Mutations at different loci can produce the same phenotype (e.g. albinism)

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Heteroplasmy

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Presence of both normal and mutated mtDNA, resulting in variable expression in mitochondrial inherited diseases.

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Uniparental disomy

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Offspring receives 2 cpoies of a chromosome from 1 parent and no copies from the other parent

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Hardy-Weinberg population genetivs

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- If a population is in Hardy-Weinberg equilibrium, then:
- Phenotype prevalence: p2 + 2pq + q2 = 1
- Allele prevelance: p + q = 1

- p and q are separate alleles. the prevalance of an X-linked recessive disease in males = q and in females = q2

- HW equilibrium assumes no mutation, no selection, random mating and no migration

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Imprinting

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- at a single locus, only 1 allele is active; the other is inactive (by methylation). deletion of the active allele leads to disease.

- Prader-Willi syndrome: deletion of normally active paternal allele. Mental retardation, obesity, hypogonadism, hypotonia.

- Angleman's syndrome: deletion of normally active maternal allele. Mental retardation, seizures, ataxia, inappropriate laughter (happy puppet).

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Modes of Inheritence

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- Autosomal Dominant: all generations affected, both male and female affected

- Autosomal Recessive: usually in only 1 generation

- X-linked Recessive: 50% of sons of heterozygous mothers are affected, no male to male transmission

- X-Linked Dominant: 50% of male or female offspring of affected mother are affected while 100% of female and no male offspring of affected males are affected.

- Mitochondrial: only transmitted through mother; variable expression due to heteroplasmy.