Genet 302 lec 12 & 13
What are the three basic steps of standard PCR analysis?
Isolate DNA, perform PCR amplification, and analyze products using agarose gel electrophoresis (AGE).
Why must PCR primers be within ~5 kb of each other in standard PCR?
Standard PCR efficiently amplifies fragments under about 5 kb; larger fragments require long-range PCR kits.
What is a limitation of agarose gel electrophoresis (AGE)?
The gel must be manually loaded and visually examined by a person.
What is the purpose of PCR when detecting chromosome rearrangements?
To determine whether a person is heterozygous or homozygous for a deletion or other rearrangement.
How is a small deletion detected using PCR?
Primers flank the deleted region; wild-type produces a larger band, mutant produces a smaller band.
What band pattern indicates a heterozygous small deletion?
Two bands: one wild-type size and one smaller mutant size.
How is a large deletion detected using PCR?
Use three primers: two outside the deletion and one inside the deleted region.
In large deletion detection, which primer pair amplifies the wild-type allele?
The two primers that flank the intact region (e.g., 1+2).
Why does the internal primer fail to amplify the deleted allele?
Because the deleted region containing the primer binding site is missing.
How can PCR products be analyzed without agarose gels?
Using fluorescent primers and capillary electrophoresis.
How does capillary electrophoresis detect PCR products?
Fluorescently labeled PCR fragments pass a detector in a capillary tube and are read by a computer as peaks.
What does the peak position in capillary electrophoresis represent?
Fragment size (migration time corresponds to length).
What is DNA methylation?
The addition of a methyl group to cytosine, forming 5-methylcytosine.
Which parental chromosome generally has more DNA methylation?
Maternal chromosomes.
What restriction enzyme is used in methylation-sensitive PCR in this lecture?
HhaI.
What sequence does HhaI recognize?
GCGC.
Why can HhaI distinguish between maternal and paternal chromosomes?
It cannot cut methylated DNA; methylated CpG sites block digestion.
Which chromosome (maternal or paternal) is cut by HhaI in the example?
Paternal chromosomes, because they are less methylated.
What is the purpose of methylation-sensitive PCR?
To determine the parental origin of a deletion or duplication.
What additional step is added in methylation-sensitive PCR?
HhaI digestion before PCR.
What are the steps for detecting parental origin of a chromosome rearrangement?
Isolate DNA, digest with HhaI, perform PCR, analyze by capillary electrophoresis.
What deletion causes Prader-Willi syndrome?
del(15)(q11q11)pat (paternal deletion).
What deletion causes Angelman syndrome?
del(15)(q11q11)mat (maternal deletion).
Why do paternal and maternal deletions cause different syndromes?
Due to genomic imprinting and differential methylation.
What duplication example is discussed in this lecture?
dup(22)(q11q11) causing Cat-eye syndrome.
How can methylation-sensitive PCR distinguish duplication parental origin?
By comparing digestion patterns after HhaI treatment.
How do you detect a small deletion using standard PCR?
Use two primers outside the deleted region and analyze by AGE.
How do you detect a large deletion using PCR?
Use two primers outside and one primer inside the suspected deletion region.
How do you detect parental origin of a deletion using PCR?
Use primers inside the region and perform methylation-sensitive digestion before PCR.
In what year was manual Sanger DNA sequencing developed and by whom?
1977 by Frederick Sanger.
In what year was automated Sanger DNA sequencing developed and by whom?
1986 by Leroy Hood.
What is the key difference between manual and automated Sanger sequencing?
Manual sequencing used separate reactions and gels; automated sequencing uses fluorescent dyes and capillary electrophoresis.
What are the three steps used in the simple Sanger sequencing example?
Isolate DNA, PCR amplify the region, perform Sanger DNA sequencing.
Why can the same PCR primers often be used for Sanger sequencing?
Because Sanger sequencing requires a primer that binds to the amplified PCR product.
In the X-linked example, why is the father hemizygous?
Males have only one X chromosome, so they carry only one allele of X-linked genes.
If a male shows only the wild-type peak pattern at an X-linked locus, what is his genotype?
Hemizygous wild-type.
If a male shows only the mutant peak pattern at an X-linked locus, what is his genotype?
Hemizygous mutant.
If a female shows overlapping peaks at one position in a Sanger trace, what is her genotype?
Heterozygous.
In the lecture example, what was the genotype of the father (I-1)?
Hemizygous for the wild-type allele.
What was the genotype of the son (II-1)?
Hemizygous for the mutant allele.
What was the genotype of the mother (I-2)?
Heterozygous.
What was the genotype of the fetus (II-2) in the example?
Affected male.
What does a single clean peak at each position in a Sanger trace indicate?
Homozygous (or hemizygous in males) sequence.
What do overlapping peaks at the same position in a Sanger trace indicate?
Heterozygosity.
In the complex ASL example, the patient was homozygous for what type of mutation?
A 7 bp deletion-insertion (7 bp delins).
What is a delins mutation?
A mutation where a segment of DNA is deleted and replaced with different nucleotides in a single event.
Would a heterozygous person show mixed overlapping peaks in the delins region?
Yes.
In the family example, did the results match standard Mendelian inheritance expectations?
No.
What alternative genetic explanation was suggested for the unexpected family result?
Uniparental disomy (UPD).
What is uniparental disomy (UPD)?
Inheritance of both copies of a chromosome from one parent.
What were alternative explanations listed besides UPD?
Spontaneous mutation, wrong gene tested, or non-paternity.
What is a sequence logo?
A graphical representation of consensus sequences in DNA, RNA, or proteins.
What does the height of letters in a sequence logo represent?
Information content (measured in bits).
What does larger letter size in a sequence logo indicate?
Higher conservation at that position.
What does smaller letter size indicate in a sequence logo?
Greater variation at that position.
What does the total height of a stack of letters represent in a sequence logo?
The overall conservation at that nucleotide or amino acid position.