Midterm 1 - Notes 4 (Part 1)

Question 1

Micro-satellites

Answer 1

Is a tract pf repetitive DNA in which certain motifs are repeated

Question 2

What 2 things could happen if we had many more markers available?

Answer 2

1. Better resolution of co-inherited blocks
   - better to pin pin point cross over thats occur in family studies (controlled)
2. Less related population could be used
   - genetic association in natural population
   - GWAS

Question 3

GWAS

Answer 3

Genome Wide Association Studies

Question 4

SNP

Answer 4

Single Nucleotide Polymorphism

Question 5

Single nucleotide polymorphism

Answer 5

A variation in a single base pair in a DNA sequence

Question 6

Whats the max amount alleles you can have in a SNP?

Answer 6

4

- but most SNPs have only 2 alleles

Question 7

Where are the 3 places polymorphism can occur?

Answer 7

1. Within coding regions
2. In non coding regions
3. In intergenic regions

Question 8

Intergenic regions

Answer 8

Is a stretch of DNA sequence located between genes

- subset of non-coding DNA

Question 9

Polymorphism

Answer 9

The condition of occurring in several different forms

Question 10

What is a huge benefit to SNPs?

Answer 10

They are extremely frequent

Question 11

SNPs are what kind of allele difference?

Answer 11

The most simplest type

Question 12

What happens if SNPs are in non coding regions? (2)

Answer 12

1. It may affect gene splicing
   - alternative gene splicing
2. Transcription factor binding
   - change the ability of a gene regulatory element from binding and changing its outcome

Question 13

What happens if SNPs are in the coding region?

Answer 13

1. 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)
2. Nonsynonymous (missense or nonsense)

Question 14

Missense

Answer 14

Change in amino acid

Question 15

Nonsense

Answer 15

Premature stop codon

Question 16

Whats the first step in SNP microarrays?

Answer 16

You have to know where the SNPs are located before starting

Question 17

What is the goal of a microarray?

Answer 17

To quantify an amount of target DNA in a mixture

- you do this parallel for many targets

Question 18

What are 3 scenarios where you can use microarrays?

Answer 18

1. Genome typing
2. Gene expression analysis
3. Epigenetics changes

Question 19

What are the 9 steps in the microarray principle?

Answer 19

1. Start out with a target sequence
2. Generate small probes that contain the actual sequence we are targeting
   - oligonucleotides
3. Immobilize the probes on a solid surface
4. On the other side we will start with DNA mixture that we want to measure the abundance of
   - eg) transcripts in the mixture
5. Prepare DNA mixture
6. Label it with fluorescence the DNA fragments in the mixture and float it over the solid surface of the microarray
7. Allowing binding to target DNA in mixture to complementary probe through hybridization
8. Wash off all unbound DNA
9. Can scan and measure the labeled spots by observing their intensity of each individual spot

Question 20

What do the results of a microarray display?

Answer 20

They display a quantification of the DNA

Question 21

What are the 6 steps in SNP arrays – Affymetrix?

Answer 21

1. Isolate genome DNA from individual
2. Fragment DNA
3. Amplify all fragments in the solution
4. Label fragments on parellel
5. Hybridize to array containing probes covering known SNP locations
   - probes set are specific to each allele at the given locus
6. Measure fluorescence for each probe spot, called genotype for each SNP

Question 22

What does shifting of one base do? (2)

Answer 22

1, Changes the position

2. Done to do a replication in the same sequence

Question 23

What are the 7 steps in SNP arrays – Illumina Infinum II

Answer 23

1. Isolate genomic DNA from the individual
   - genomic DNA target SNP
2. Fragment DNA
3. Amplify all fragments in solution by PCR
4. Hybridize the bead-array containing probes covering known SNP locations
   - probe ends one base before SNP location (open 5’-end)
5. Extend probe by one base only (SNP location) using differently labelled ddNTPs and DNA polymerase
6. Measure dual-colour fluorescence for each probe bead, called genotype for each SNP
7. DNA polymerase can then add the next base, but only it it is int eh correct order/ complement

Question 24

PCR

Answer 24

Polymerase Chain Reaction

Question 25

What are ddNTPs?

Answer 25

Are essentially stop base codons to ensure only one base is added
- you get a strong genomic binding

Question 26

How many spots can a single array have?

Answer 26

Millions of spots

Question 27

What does the number of SNPs covered depend on?

Answer 27

Depends on the number of SNPs known at the time of array design

Question 28

How many markers does Affymetrix human array have?

Answer 28

1.8 million markers (including > 900,000 SNPs)

Question 29

How many markers does Illumina human bead array have?

Answer 29

> 1.2 million markers (including > 1,100,000 SNPs)