Day 10.3 Genetics Flashcards
What is the purpose of PCR
Amplify a desired fragment of DNA (eg an allele)
What are the steps of PCR?
- Denaturation - heat the DNA to separate the 2 strands
- Annealing- During cooling, premade DNA primers anneal to a specific seq on the strand to be amplified
- Elongation - heat-stable DNA polymerase replicates the DNA seq after each primer –> then have 2 copies
- Repeat a lot for amplification.
Agarose gel electrophoresis
Used to separate PCR products of different sizes. Negative end (cathode) has wells, and DNA is negatively charged, so it runs toward positive end (anode). Smaller molecules (low molecular weight) travel further bc it is easier for them to get through the gel Results are compared to (known) DNA ladder.
What is unique about the primers used in PCR?
They’re DNA primers (vs the normal RNA primers)
How is PCR for proteins different?
Proteins can be positive or negative, so the wells are in the middle, so that they can travel in either direction. So, PCR identifies both charge and size (smaller travels further)
Southern blot
Southern = DNA sample (and DNA probe)
DNA sample is electrophoresed on gel and txfrd to a filter. Filter is soaked in a denaturant, and then exposed to a labeled DNA probe.
Labeled probe binds to the DNA, making double stranded DNA.
Double strand is seen when the filter is exposed to film.
Northern blot
Northern = RNA (and DNA probe)
RNA is electrophoresed on a gel and then transferred to a filter.
Filter is soaked in denaturant and exposed to DNA probe. DNA binds to RNA and double stranded RNA-DNA hybrid is seen when filter is exposed to film.
Western blot
Western = protein sample (Ab probe)
Sample protein is separated via electrophoresis and transferred to a filter. Labeled Ab is used to bind to the protein.
Southwestern blot
Southwestern - DNA binding proteins (transcription factors) are the sample. They are idenitified using labeled oligonucleotides as the probes.
What is an oligonucleotide?
A short sequence of nucleic acids (single stranded)- e.g. a primer is an oligonucleotide.
What blotting procedure is used to determine gene expression?
Northern (so you can look at the RNA sequence that is transcribed) Northern = RNA
What is a microarray?
Lots of nucleic acid seq’s are arranged in a certain order in grids on a glass/silicon chip.
RNA or DNA probes are hybridized to the chip, chip is scanned to detect the relative amts of binding.
What is microarray used for?
To look at gene expression lvls or to detect SNPs- single nucleotide polymorphisms
For genotyping, forensics, predisposition to dz, cancer mutation, genetic linkage analysis
ELISA
Rapid test for Ag-Ab reactivity.
2 ways to do it:
1. Use a labeled test Ag to see if the pt’s immune system recognizes it (e.g. use HBsurfAg to see if pt has Ab against it; HIV Ag to see if pt has anti-HIV Ab)
2. Use a labeled test Ab to see if the pt has Ag present
The test substance (either Ag or Ab) is coupled to a color-generating enz, so if the target substance is present in the sample, the solution will have an intense color rxn, indicating a positive result.
What is the sensitivity and specificity of the ELISA test?
Both are 100%
But both FP and FN results do occur.
FISH
Fluorescent DNA or RNA probe binds to specific gene site of interest.
Used for specific localization of genes and direct visualization of anomalies (like microdeletions) at a molecular level
Used when deletion is too small to be visualized by karyotyping.
Fluorescence - gene is present. (If no fluor, it is not)
Karyotyping
Done in Metaphase.
Chromosomes are stained, ordered, numbered by morphology, size, arm-length ratio, banding pattern.
Can be performed on sample of blood, bone marrow, amniotic fluid, placental tsu.
Used to dx chromosomal imbalances- autosomal trisomies, sex chr disorders
Cloning methods
- Isolate eukaryotic mRNA of interest
- Expose the mRNA to a reverse transcriptase, to make cDNA
- Insert the cDNA fragments into bacterial plasmids w abx-resistant genes
- Put the bacteria on Abx plate- the ones that survive have the cDNA. Make a cDNA library.
Cloning: what is cDNA
DNA that lacks introns
Cloning: how is the DNA able to be inserted into plasmids?
Restriction enz cleave DNA at 4bp to 6bp palindromic sequences, which allows them to be inserted.
What is Sanger DNA sequencing?
Dideoxynucleotides halt DNA polymerization at each base, so sequences of various lengths are generated (and overall, all of the sequences cover the entire original sequence)
Terminated fragments are electrophoresed and the original sequence is deduced from matching up the overlaps.
What is knock-in?
Inserting a gene.
Constitutive: Gene insertion is random (into any place in the genome) so it could be inserted in the middle of something important and make it non-fnl
Conditional: insertion is targeted thru homologous recombination w the mouse gene. This is better.
What is knock-out?
Removing a gene
Conditional: targeted deletion of gene thru homologous recombination w the mouse gene.
In mouse model systems, how can the gene be manipulated at a specific devtl point?
Inducible cre-lox system w an abx-controlled promoter.
Eg to study a gene whose deletion causes embryonic lethality.
What is RNAi?
dsRNA that is complementary to a mRNA seq of interest is synth’d.
When it’s transfected into cells, the dsRNA separates and promotes degredation of the target mRNA, knocking down gene expression. (no mRNA means no proteins)
Codominance
Neither of 2 alleles is dominant, each has an effect
Ex: Blood groups A, B, AB
Variable expression
The nature and severity of a phenotype can vary from one individual to another even if they both have the same dz mutation
Ex: 2 pts w neurofibromatosis or tuberous sclerosis can have different dz severity
Pleiotropy
One gene has more than one effect on an individual’s phenotype
Ex: PKU causes seemingly unrelated sx - MR, hair and skin changes
Incomplete penetrance
Not all individuals with mutant genotype show the mutant phenotype
THis is why a dz can “skip generations”- mutant gene is always there, but may not show in phenotype.
Imprinting- how does it affect phenotype?
Differences in phenotype depend on whether mutation is in paternal or maternal gene.
Ex: Prader-Willi and Angelman’s syndromes are both d/t inactivation or deletion on Chr 15.
What is imprinting?
Imprinting is silencing of one allele (inactivated by methylation). Therefore, at that locus, only the other allele that is not imprinted is active. If the one active allele (the one that is not imprinted) is defective/deleted, then there is dz.
Prader-Willi syndrome
Ex of imprinting. Mom’s allele was imprinted (silenced), and the normally active Paternal allele is deleted.
Prader = Paternal deletion on Chr 15
Px in infancy w hypotonia, poor feeding, almond eyes and downward mouth.
Sx: MR, obese and hyperphagic, hypotonia, short stature (partial GH deficiency), bhvr disorders, and hypogonadotrophic hypogonadism –>genital hypoplasia, childhood osteoporosis, and delayed menarche
Angelman’s syndrome
Ex of imprinting. Dad’s allele was imprinted (silenced), and the normally active Maternal allele is deleted.
angelMan’s = Maternal deletion on Chr 15
Sx: MR, seizures, ataxia, inappropriate laughter (happy puppet)
What is the difference bt variable expression and pleiotropy?
Variable exprsn means the nature and severity of phenotype can vary bt individuals
Pleiotropy means that one gene can have more than one affect on the phenotype (eg can affect both brain and skin)
Anticipation
Severity of disease worsens w successive generations
Or, age of onset is earlier in successive generations
Ex trinucleotide repeat dz like huntington’s
Loss of heterozygosity
Must lose both alleles before there is an effect.
Eg if a pt inherits a mutation in a tumor suppressor gene, the pt is fine- unless there is also a mutation/deletion in the complementary allele –> then, cancer. (Two-hit hypoth)
Ex: retinoblastoma, p53 mutations
Note: this is NOT true for oncogenes- they have a gain of fn mutation, so only need one mutated.
Dominant negative mutation
Mutation exerts a dominant effect- so a heterozygote will make a non-fnl altered protein that also prevents the normal gene product from fn’g
Ex: mutation of Tx factor in its allosteric site. The non-fn’g mutant can still bind DNA, preventing the wild-type Rx factor from binding. (i don’t think this is the best ex)
Linkage disequilibrium
Tendency for certain alleles in the same gene to occur together more often than they would by chance.
Measured in an entire population (not in a family) and often varies in different populations.
Mosaicism
Occurs when cells in the same body have different genetic makeups.
If it’s germ-line mosaic, it will not affect the adult which has the mosaicism, but can produce dz in that person’s offspring.
Examples of mosaicism
Lyonization- random X inactivation in some cells in females
Mutation in the embryonic precursor of the bone marrow stem cell –>hematologic mosaicism
Chimeric pt- 2 zygotes that fuse, so 2 DNAs in one pt
Locus heterogeneity
Mutations at different loci can produce the same phenotype
Ex: several different things produce marfan’s habitus:
marfan’s syndrome
MEN 2B
homocystinuria (can’t metabolise methionine)
Heteroplasmy
Presence of both normal and mutated mtDNA, resulting in variable expression in mitochondrial inherited dz
Uniparental disomy
Offspring gets 2 copies of chromosome from 1 parent, and no copies from other parent
Can look like imprinting.
Hardy-Weinberg eqlbm
Disease prevalence:
p^2 + 2pq + q^2 = 1
Allele prevalence:
p + q = 1
2pq = heterozygote prevalence p^2 = pp prevalence, q^2 = qq prevalence (so p^2 + q^2 is the prevalence of ALL homozygotes)
What is the prevalence of an X-linked recessive dz in males and in females, using Hardy-Weinberg?
Males: q
Females: q^2
What are the assumptions of Hardy-Weinberg law?
- No mutations at the locus
- No selection for any of the genotypes at the locus
- Completely random mating
- No migration
If 1% of all individuals have an auto-recessive dz, what % of alleles are not mutant?
Auto-recess means that only pp will have it. So prevalence of pp is p^2 = 1%.
So, p is sq rt of 0.01, which is 0.1
Since p = 0.1,
q = 0.9 (since p + q = 1 is allele prevalence)
So, 90% of alleles are NOT mutant
(and 10% are)
Also, 1 % are pp, 18% are pq, and 81% are qq
Autosomal dominant inheritance
Only one allele needs to be mutated to cause dz
Often d/t defects in structural genes
Many generations, both male and female affected
50% of offspring affected (either sex)
Often pleiotropic (meaning the one gene has more than one effect on phenotype), and often present after puberty. (Huntington’s)
Fam hx is crucial to dx.
Autosomal recessive inheritance
Phenotype only present when you have both copies of the allele.
Often d/t enz deficiencies
Usu only seen in one generation (unless incest)
Commonly more severe than auto-dom, pts often px in childhood
If both parents are carriers (Rr), then 25% chance that offspring will get it.
X-linked recessive
Males are affected more severely bc they only have one X
Sons of heterozyg mom get it 50% of the time
Females of heterozyg mom are carriers 50% of time.
Homozyg females are affected (dad had it and mom was a carrier)
X-linked dominant
Transmitted thru both parents
ALL female offspring of an affected father have it.
An affected mother can pass it on to either male or female offspring.
Hypophosphatemic rickets
(aka vit d resistant rickets)
X-linked dom
Increased phosphate wasting at proximal tubule
Results in rickets-like presentation
Mitochondrial inheritance
Inheritance is thru mother ONLY.
ALL offspring of affected females have dz.
There is variable exprsn in the population d/t heteroplasmy (presence of both normal and mutated mito DNA)
List 3 mitochondrial inheritance defects
- Mitochondrial myopathy - see ragged-red musc fibers on biopsy
- Leber’s hereditary optic neuropathy - degeneration of retinal ganglion cells and axons, leads to acute loss of central vision
- Leigh syndrome - subacute sclerosing encephalopathy
Females have a 50% chance of being a carrier
X-linked recessive
Usu only seen in one generation
Auto-recessive
If a male is affected, all of his daughters will be affected
X-linked dom
