Genet 302 tech review
What is the purpose of PCR?
In vitro exponential amplification of a small region of DNA.
What natural process is PCR based on?
DNA replication.
What happens to double-stranded DNA when heated?
It denatures into single strands.
What happens during renaturation of DNA?
Complementary single-stranded DNA pieces pair.
Why is Taq polymerase used in PCR instead of E. coli DNA polymerase?
Taq polymerase withstands boiling temperatures and functions optimally at 72°C.
What is the optimal temperature for E. coli DNA polymerase?
37°C.
What is the optimal temperature for Taq polymerase?
72°C.
What are oligonucleotides (oligos)?
Synthetic single-stranded DNA molecules ~20 nucleotides long.
What is the role of primers in PCR?
They bind complementary DNA and serve as starting points for DNA synthesis.
What must you know before designing PCR primers?
The DNA sequence at both ends of the region to be amplified.
What is the approximate maximum size of DNA that standard PCR can amplify?
About 5 kb.
What components are required in a PCR reaction?
Template DNA, Taq DNA polymerase, forward primer, reverse primer, and dNTPs.
What machine is used to run PCR?
A thermocycler.
What are the three steps of one PCR cycle?
Denaturation, annealing, and extension.
At what temperature does denaturation occur?
~95°C.
At what temperature does annealing typically occur?
~55°C (varies depending on primer).
At what temperature does extension occur?
~72°C.
How many cycles are typically run in a PCR reaction?
About 30 cycles.
What is the theoretical amplification pattern of PCR?
Exponential doubling each cycle (2^n).
After 30 cycles, what types of DNA are present in the tube?
Original template, unused primers/dNTPs, variable-length DNA, and lots of constant-length target DNA.
Which PCR products are visible on an agarose gel?
The constant-length target DNA.
Where do primers end up in the final PCR product?
They become incorporated at the ends of the amplified DNA fragment.
If a region is 800 bp and each primer is 20 bp, what is the final PCR product length?
840 bp.
What is one major use of PCR?
Amplifying DNA for sequencing.
How can PCR detect expanded repeats in a gene?
Expanded repeats produce longer PCR products visible on a gel.
What gene example was used for repeat expansion detection?
FMR1.
What distinguishes FMR1+ from FMR1− alleles?
FMR1− has >200 repeats; FMR1+ has <200 repeats.
What are the steps to determine genotype using PCR?
Isolate DNA, perform PCR, run agarose gel electrophoresis (AGE).
What does AGE stand for?
Agarose gel electrophoresis.
What determines PCR band size on a gel?
The distance between primers plus any insertions or repeat expansions.
What is the purpose of FISH?
To identify specific genes or chromosomes in interphase or metaphase cells using fluorescent probes.
What molecule is commonly used to stain DNA blue in FISH?
DAPI, a synthetic molecule with high affinity for DNA.
What are FISH hybridization probes typically made from?
BAC (bacterial artificial chromosome) clones.
What is random prime labeling used for in FISH?
To incorporate fluorescently labeled nucleotides into BAC DNA to create a probe.
What is an example of a fluorescent nucleotide used in FISH probes?
Cy3-dCTP.
If a probe is made from one BAC targeting a unique region, how many signals appear in a normal diploid cell?
Two signals, one on each homologous chromosome.
What is the approximate size of DNA in a BAC clone?
~100,000 base pairs.
What is the purpose of centromere probes in FISH?
To count chromosomes and detect aneuploidy.
What FISH result indicates Down syndrome?
Three signals for chromosome 21 (47,XX,+21).
What is the purpose of gene-specific FISH probes?
To determine the presence or deletion of a specific gene or region.
What FISH result indicates Cri-du-chat syndrome?
Loss of one 5p15.2 signal (46,XX,del(5)(p15.2)).
What is chromosome painting used for?
To visualize entire chromosomes and detect translocations.
What is multicolour FISH?
Using multiple fluorescent probes simultaneously to distinguish chromosomes.
In what year was manual Sanger sequencing developed?
1977.
In what year was automated Sanger sequencing developed?
1986.
In what year did next-generation sequencing become widespread?
2008.
What is the function of a ddNTP in Sanger sequencing?
It terminates DNA synthesis because it lacks a 3′ OH group.
Why does a ddNTP terminate DNA synthesis?
Because without a 3′ OH, no additional nucleotide can be added.
What components are required in a Sanger sequencing reaction?
Template DNA, one primer, Taq polymerase, normal dNTPs, and fluorescent ddNTPs.
How many cycles are typically run in automated Sanger sequencing?
About 25 cycles.
What separates DNA fragments in automated Sanger sequencing?
Capillary electrophoresis.
How are bases detected during capillary electrophoresis?
A laser excites fluorescent dyes and a detector records colored peaks.
What is a chromatogram?
A graph of colored peaks representing the DNA sequence.
Approximately how many high-quality nucleotides are obtained per Sanger read?
About 700 nucleotides.
What causes PKU?
Inability to convert phenylalanine to tyrosine due to PAH deficiency.
Which enzyme is deficient in PKU?
Phenylalanine hydroxylase (PAH).
Who discovered excess phenylalanine causes PKU?
Ivar Følling in 1934.
What was the Guthrie test used for?
Detecting excess phenylalanine in newborn blood.
How did the Guthrie test work?
Newborn blood was used to feed bacteria that grow only if phenylalanine is present.
What is the modern method for newborn PKU screening?
Tandem mass spectrometry.
What percentage of breast cancer cases are non-hereditary?
90–95%.
What causes most non-hereditary breast cancers?
Somatic mutations such as in TP53.
What percentage of breast cancer cases are hereditary?
5–10%.
Which genes are commonly mutated in hereditary breast cancer?
BRCA1 and BRCA2.
What is the inheritance pattern of BRCA mutations?
Autosomal dominant.
What is the approximate cancer risk by age 70 for females with BRCA1 mutations?
~83%.
What is the purpose of GWAS?
To map gene(s) responsible for a phenotype using SNP markers across the genome.
What does GWAS stand for?
Genome-wide association study.
What technology is commonly used in GWAS?
DNA microarrays.
What is plotted in a Manhattan plot?
-log10(p-value) versus chromosomal position.
What does a single strong peak in a Manhattan plot suggest?
One gene strongly associated with the phenotype.
What do multiple peaks suggest?
Multiple genes contribute to the phenotype.
What are SNPs?
Single nucleotide polymorphisms that serve as genetic markers.
Can SNPs cause missense mutations?
Yes.
Can SNPs cause frameshift mutations?
No, a single nucleotide substitution does not cause a frameshift.
What was the older method for gene mapping before GWAS?
RFLP mapping.
Why was RFLP mapping inefficient?
It was slow, expensive, radioactive, and technically demanding.