Topic 4: Techniques for Detecting Variation at DNA Level

Question 1

What advantages do methods of analyzing DNA variation confer?

Answer 1

1. You can use the same method to examine all regions of DNA (you can examine neutral variation)
2. More variation exists at the DNA level than at the protein level (due the redundancy of the human genome)

Question 2

What are the two types of mutations at the DNA level?

Answer 2

1. Large scale: deletions, duplications, inversions, insertions, translocations
2. Small scale: base substitutions, insertions, and deletions (SNPS)

Question 3

What are most mutations classified as? How often do they occur?

Answer 3

Most mutations are substitutions (SNPs), and they occur once every 300 bp in a human genome

Question 4

What are the 8 considerations when picking a type of genetic marker?

Answer 4

1. Mutation rate
2. Abundance in genome
3. Information content per locus
4. Ease and knowledge of genotyping
5. Previous knowledge of genome of interest
6. Codominant vs dominant DNA
7. Allelic vs non-allelic data
8. Phased vs unphased data

Question 5

What are 4 ways of detecting DNA base substitution variation in homologous pieces of DNA?

Answer 5

Sanger sequencing
Restriction enzyme site variation
SNP detection
Whole genome DNA sequencing

Question 6

What two methods do you need when using each DNA variation detection method?

Answer 6

You need a method of identifying a region of the genome, and then a method of assaying it

Question 7

What is gel electrophoresis?

Answer 7

DNA molecules, when exposed to positive pole, will move down a gel towards the positive (due to negative charged phosphates)
The shape of the molecules determines the rate of migration
Long fragments will remain at the top of the gel and shorter fragments will migrate faster towards the bottom of the gel
DNA is run on either a polyacrylamide or agarose gel

Question 8

What are the three ways to visualize DNA on a gel?

Answer 8

Ethidium bromide: intercalates between the bases of nucleic acids and fluoresces red/orange in UV light, you can see ALL the DNA on a gel
SYBR-green: forms a complex with DNA that absorbs blue light and emits green light, visualizes all DNA
Radioactivity: label DNA with radioactivity then use autoradiography on a x-ray film, ONLY shows the labelled DNA, or DNA hybridized to a probe

Question 9

What are restriction enzymes?

Answer 9

These are enzymes that recognize and cur double stranded DNA at specific sequences which are palindromic. The sites at which these enzymes cut can be polymorphic! They can leave blunt ends (cut the same site on both strands) or “sticky ends” and cut unequally (also called staggered ends)

Question 10

What is an RFLP?

Answer 10

Restriction fragment length polymorphism, this is variation in the sites recognized by restriction enzymes. It can either make a new restriction site where there previously was not one, or it can change an existing restriction site therefore it is not longer one. They are much cheaper and easier to assess than sequencing

Question 11

What are the three techniques to obtain a restriction map for a DNA fragment?

Answer 11

1. Perform single or multiple digests (one or more types of restriction enzymes with different cut sites)
2. Partial digests of DNA fragment with 5’ end labelling (sometimes the enzyme cuts and sometimes it does not, leading to different length fragments showing where the cut sites are)
3. DNA sequencing of the fragment and look for the cut site sequences

Question 12

TRUE OR FALSE:
RFLP data is codominant and allelic

Answer 12

TRUE

Question 13

What is a major difference between a single/multiple digest and a partial digest?

Answer 13

In a single/multiple digest, the entire fragment is cut at all sites, but in a partial digest is it not always cut. Therefore the lengths of the fragment will add up to the total length of the DNA strand in a single/multiple digest, but in a partial digest, only the fragment that is cut and attached to the 5’ labelled end is shown thus the lanes will not add up to the total length of the fragment.

Question 14

How else can you predict the location of restriction sites?

Answer 14

Use sequence data to find the location, by looking at the bases, this is cheaper and more efficient for a diagnostic site

Question 15

What is hybridization?

Answer 15

Denature double stranded DNA to make single stranded DNA, then anneal a probe or PCR primers to the single stranded DNA, and finally you have hybrid double stranded DNA

Question 16

What are DNA probes? What method of detecting variation are they often used in?

Answer 16

DNA probes are labeled DNA that can be used for a specific target sequence. It can be designed from the sequence of a related species, or from a random cloned fragment of the same species. They are often used in the annealing step of hybridization.W

Question 17

What is a common way to label DNA using radioactivity?

Answer 17

Use nucleotides labeled with radioactive 32P and visualize using an x-ray film (autoradiography)

Question 18

Does DNA have to be labelled with radioactivity? What are two other methods of labeling?

Answer 18

NO, they can also be labeled with non-radioactive substances such as biotin and fluorescent dyes.

Question 19

What are the steps in making a southern blot?

Answer 19

You use restriction enzymes to cut DNA then run it on gel electrophoresis, and then you use a buffer which transfers DNA onto a nitrocellulose paper and you then denature DNA and hybridize with labeled DNA or a probe, and then visualize it on autoradiography. On the southern blot, you ONLY see the DNA that is hybridized to the probe.