Molecular Genotyping Lecture Flashcards
Mendelian segregation of trait differences in humans is _____
rare
Why can’t you set up crosses for humans?
because generation time is LONG, and offspring number is low.
What does a genetic cross or pedigree help you study?
1) segregation of a phenotype. 2) segregation of any genotype 3) map genetic distance with molecular markers 4) studying both genotypes and phenotypes.
point of a genetic cross or a pedigree is to study both genotypes and phenotypes. But why do we care about studying both genotypes and phenotypes?
it lets you look for linkage between the phenotype and the genotype of interest.
So how do you exactly look for a genotype that is responsible for a phenotype occurring in the individual?
You need: 1) A family and their DNA where some people have the disease and some don’t. 2) The ability to track which child got which chromosome from which parent.
A change in the DNA sequence does not need to _______ the phenotype of the organism for us to be able to track it.
does not need to change
an important property of molecular markers is that ____
they can segregate according to mendelian rules
what can molecular markers be used for?
1) one can link the molecular marker to the human disease 2) it can be then used as a starting point to find the relevant DNA sequence changes that caused the disease.
We learned that change in the DNA sequence does not need to change the phenotype of the organism for us to be able to track them. In such a case, how do we find the relevant DNA sequence variation?
we just learned Molecular Markers
What is SNP?
Single Nucleotide Polymorphism. Which means change in a single nucleotide. eg. instead of G in a sequence we have A.
What is polymorphic site in SNP?
The position of nucleotide variation
How can SNPs be genotyped?
PCR analysis of restriction single site altering SNPs
DNA microarrays for millions of SNPs
Whole genome sequencing for millions of SNPs
What process will you use to distinguish DNA fragments according to size?
Gel Electrophoresis
How do you conduct Gel electrophoresis?
linear DNA fragments migration distance through gel depends on size.
After electrophoresis visualize DNA fragments by staining gel with fluorescent dye and photograph gel under UV light.
Determine size of unknown fragments by comparison to migration of DNA markers of known size.
How does the sickle cell mutation eliminate an MstII restriction site?
1) PCR of the region containing SNP produces fragments from both alleles normal and sickle cell.
2) Restriction enzyme (MstII) application to the PCR product.
AA
What is a microsatellite?
microsatellite is a simple sequence repeat with a highly variable number of short sequence repeats ranging from 2 - 4 nucleotides long.
eg. CACACACACACACACA
How can you determine SSR polymorphis by PCR and Gel electrophoresis?
What is the genotype of all 6 people shown here?
How do you identify the disease genotype?
because all affected children have only one band in common and the father also has that band.
How can a microsatellite locus show linkage to a disease gene?
If you look at the microsatellite sizes in a) you will notice that in b) the M’ - M’’’’ are arranged in size from longest to shortest. then you identify the diseased allele band and see which people is it expressed in. Those people are the affected individuals.
remember: parental genotypes serve as base line, then children are analyzed the same way but compared to parental to find the answer.
Most common DNA markers used as molecular markers and why?
1) Short sequence length polymorphisms
2) other DNA polymorphisms
Occur the most in the human genome.
How do you map and clone a disease locus?
