Molecular markers

Question 1

What are restriction fragment length polymorphisms in actuality?

Answer 1

Snip SNPs - single nucleotide polymorphisms that are in the context of a restriction enzyme cut site

Question 2

Are RFLPs highly polymorphic?

Answer 2

No, 2 alleles. Presence or absence of a cut site

Question 3

How do we detect RFLPs?

Answer 3

Southern blot or PCR

Question 4

What would homozygotes and heterozygotes look like for the different alleles of RFLPs?

Answer 4

Homozygote for presence of site: two smaller bands
Homozygote for absence of site: one larger band
Heterozygote: 3 bands

Question 5

What’s another name for a minisatellite?

Answer 5

VNTRs, variable number tandem repeats

Question 6

Are minisatellites highly polymorphic?

Answer 6

Yes, and with high degree of heterozygosity

Question 7

What is the problem with using RFLPs as a marker?

Answer 7

There’s not a lot of them. Only a small subset of SNPs are snip SNPs

Question 8

How do we detect minisatellites?

Answer 8

Southern blot. Use an enzyme that cuts outside the repeat to find the size of the allele and use the repeat as a probe

Question 9

Why can’t we do PCR on minisatellites?

Answer 9

Too big

Question 10

Are minisatellites spread through the genome?

Answer 10

No, they’re localized to subtelomeric regions of chromosomes

Question 11

What are the problems with using minisatellites as a marker?

Answer 11

1. Detection can only be done by Southern blot, which is time consuming and tedious
2. Are only in subtelomeric regions

Question 12

What are two other names for microsatellites?

Answer 12

Simple sequence repeats (SSRs) and simple tandem repeats (STRs)

Question 13

Are microsatellites polymorphic?

Answer 13

Yes and high heterozygosity

Question 14

How do we detect microsatellites?

Answer 14

PCR using primers that flank on either side of the repeat and polyacrylamide gel electrophoresis

Question 15

Are microsatellites spread through the genome?

Answer 15

Yes

Question 16

What is the problem with using microsatellites as a marker?

Answer 16

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

Question 17

What is multiplex PCR?

Answer 17

A handy way to save time by intentionally amplifying multiple products in the same PCR reaction

Question 18

How do we tell products from different reactions apart in multiplex PCR?

Answer 18

1. Different sizes

2. Differentially fluorescently labelled primers for each reaction

Question 19

What are SNPs?

Answer 19

Single nucleotide polymorphisms - positions in the genome where the nucleotide there can vary

Question 20

Are SNPs highly polymorphic?

Answer 20

Ish. Can have up to 4 alleles, but most SNPs have 2

Question 21

Are SNPs distributed throughout the genome?

Answer 21

They’re everywhere and there’s about 12 million of them

Question 22

How do we detect SNPs?

Answer 22

SNP Chips using hybridization

Question 23

What is a SNP chip?

Answer 23

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

Question 24

How do we prepare a sample for a SNP chip?

Answer 24

1. Isolate DNA from a patient
2. Fragment DNA with sonication, mechanical force, restriction enzymes and denature it into ssDNA
3. Label the fragments with a fluorophore or biotin

Question 25

How do we do a SNP chip experiment?

Answer 25

1. Add prepared patient DNA to the chip under high stringency conditions
2. Add fluorochrome labelled strepavidin and detect the biotin probe on the annealed patient DNA
3. Detect the labelled strepavidin

Question 26

What would the results of a SNP chip experiment look like for a homozygote and for a heterozygote?

Answer 26

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

Question 27

What are the most commonly used markers today?

Answer 27

SNPs and microsatellites

Question 28

How did we build these high density reference maps with SNPs?

Answer 28

CEPH families