Midterm 1 - Notes 4 (Part 1) Flashcards
Micro-satellites
Is a tract pf repetitive DNA in which certain motifs are repeated
What 2 things could happen if we had many more markers available?
- Better resolution of co-inherited blocks
- better to pin pin point cross over thats occur in family studies (controlled) - Less related population could be used
- genetic association in natural population
- GWAS
GWAS
Genome Wide Association Studies
SNP
Single Nucleotide Polymorphism
Single nucleotide polymorphism
A variation in a single base pair in a DNA sequence
Whats the max amount alleles you can have in a SNP?
4
- but most SNPs have only 2 alleles
Where are the 3 places polymorphism can occur?
- Within coding regions
- In non coding regions
- In intergenic regions
Intergenic regions
Is a stretch of DNA sequence located between genes
- subset of non-coding DNA
Polymorphism
The condition of occurring in several different forms
What is a huge benefit to SNPs?
They are extremely frequent
SNPs are what kind of allele difference?
The most simplest type
What happens if SNPs are in non coding regions? (2)
- It may affect gene splicing
- alternative gene splicing - Transcription factor binding
- change the ability of a gene regulatory element from binding and changing its outcome
What happens if SNPs are in the coding region?
- Synonymous (silent mutations)
- one base pari can change the sequence and have no effect, but it can also change and create a stop codon (more common to have no change) - Nonsynonymous (missense or nonsense)
Missense
Change in amino acid
Nonsense
Premature stop codon
Whats the first step in SNP microarrays?
You have to know where the SNPs are located before starting
What is the goal of a microarray?
To quantify an amount of target DNA in a mixture
- you do this parallel for many targets
What are 3 scenarios where you can use microarrays?
- Genome typing
- Gene expression analysis
- Epigenetics changes
What are the 9 steps in the microarray principle?
- Start out with a target sequence
- Generate small probes that contain the actual sequence we are targeting
- oligonucleotides - Immobilize the probes on a solid surface
- On the other side we will start with DNA mixture that we want to measure the abundance of
- eg) transcripts in the mixture - Prepare DNA mixture
- Label it with fluorescence the DNA fragments in the mixture and float it over the solid surface of the microarray
- Allowing binding to target DNA in mixture to complementary probe through hybridization
- Wash off all unbound DNA
- Can scan and measure the labeled spots by observing their intensity of each individual spot
What do the results of a microarray display?
They display a quantification of the DNA
What are the 6 steps in SNP arrays – Affymetrix?
- Isolate genome DNA from individual
- Fragment DNA
- Amplify all fragments in the solution
- Label fragments on parellel
- Hybridize to array containing probes covering known SNP locations
- probes set are specific to each allele at the given locus - Measure fluorescence for each probe spot, called genotype for each SNP
What does shifting of one base do? (2)
1, Changes the position
2. Done to do a replication in the same sequence
What are the 7 steps in SNP arrays – Illumina Infinum II
- Isolate genomic DNA from the individual
- genomic DNA target SNP - Fragment DNA
- Amplify all fragments in solution by PCR
- Hybridize the bead-array containing probes covering known SNP locations
- probe ends one base before SNP location (open 5’-end) - Extend probe by one base only (SNP location) using differently labelled ddNTPs and DNA polymerase
- Measure dual-colour fluorescence for each probe bead, called genotype for each SNP
- DNA polymerase can then add the next base, but only it it is int eh correct order/ complement
PCR
Polymerase Chain Reaction
What are ddNTPs?
Are essentially stop base codons to ensure only one base is added
- you get a strong genomic binding
How many spots can a single array have?
Millions of spots
What does the number of SNPs covered depend on?
Depends on the number of SNPs known at the time of array design
How many markers does Affymetrix human array have?
1.8 million markers (including > 900,000 SNPs)
How many markers does Illumina human bead array have?
> 1.2 million markers (including > 1,100,000 SNPs)
