Molecular markers Flashcards
What are restriction fragment length polymorphisms in actuality?
Snip SNPs - single nucleotide polymorphisms that are in the context of a restriction enzyme cut site
Are RFLPs highly polymorphic?
No, 2 alleles. Presence or absence of a cut site
How do we detect RFLPs?
Southern blot or PCR
What would homozygotes and heterozygotes look like for the different alleles of RFLPs?
Homozygote for presence of site: two smaller bands
Homozygote for absence of site: one larger band
Heterozygote: 3 bands
What’s another name for a minisatellite?
VNTRs, variable number tandem repeats
Are minisatellites highly polymorphic?
Yes, and with high degree of heterozygosity
What is the problem with using RFLPs as a marker?
There’s not a lot of them. Only a small subset of SNPs are snip SNPs
How do we detect minisatellites?
Southern blot. Use an enzyme that cuts outside the repeat to find the size of the allele and use the repeat as a probe
Why can’t we do PCR on minisatellites?
Too big
Are minisatellites spread through the genome?
No, they’re localized to subtelomeric regions of chromosomes
What are the problems with using minisatellites as a marker?
- Detection can only be done by Southern blot, which is time consuming and tedious
- Are only in subtelomeric regions
What are two other names for microsatellites?
Simple sequence repeats (SSRs) and simple tandem repeats (STRs)
Are microsatellites polymorphic?
Yes and high heterozygosity
How do we detect microsatellites?
PCR using primers that flank on either side of the repeat and polyacrylamide gel electrophoresis
Are microsatellites spread through the genome?
Yes
What is the problem with using microsatellites as a marker?
They expand and contract during replication because of strand slippage, and PCR involves a lot of replication. Results in a lot of small stutter bands from strand slippage
What is multiplex PCR?
A handy way to save time by intentionally amplifying multiple products in the same PCR reaction
How do we tell products from different reactions apart in multiplex PCR?
- Different sizes
2. Differentially fluorescently labelled primers for each reaction
What are SNPs?
Single nucleotide polymorphisms - positions in the genome where the nucleotide there can vary
Are SNPs highly polymorphic?
Ish. Can have up to 4 alleles, but most SNPs have 2
Are SNPs distributed throughout the genome?
They’re everywhere and there’s about 12 million of them
How do we detect SNPs?
SNP Chips using hybridization
What is a SNP chip?
Silicon computer chips with single stranded 25 nucleotide oligomers probes printed on them. Would have about 500 000 different SNPs on there and will have 1 spot per allele
How do we prepare a sample for a SNP chip?
- Isolate DNA from a patient
- Fragment DNA with sonication, mechanical force, restriction enzymes and denature it into ssDNA
- Label the fragments with a fluorophore or biotin
How do we do a SNP chip experiment?
- Add prepared patient DNA to the chip under high stringency conditions
- Add fluorochrome labelled strepavidin and detect the biotin probe on the annealed patient DNA
- Detect the labelled strepavidin
What would the results of a SNP chip experiment look like for a homozygote and for a heterozygote?
If a patient is homozygous for a SNP allele at a certain locus, only one spot will fluoresce. If they’re heterozygous, both spots for that locus will fluoresce
What are the most commonly used markers today?
SNPs and microsatellites
How did we build these high density reference maps with SNPs?
CEPH families
