Lecture 6: Sanger and sequencing

Question 1

Define tandem repeats

Answer 1

Repeats in DNA that are directly adjacent to each other

Question 2

Define dispersed repeats

Answer 2

repeats found in varying regions of the genome

Question 3

Walter Fiers was able to do what in 1972?`

Answer 3

He sequenced the first protein coding gene, from RNA bacteriophage MS2

Question 4

What was first genome to be sequenced?

Answer 4

RNA bacteriophage MS2

Question 5

Describe Sanger chain termination method (3 points)

Answer 5

1. the synthesis of DNA takes place, complementary to the “template” of interest
2. Upon addition of dideoxynucleotide, the strand will stop synthesis
3. This addition will take place at different points in the template, yielding a number of fragments that can be pieced together

Question 6

How many parallel sets of reactions take place in Sanger chain termination method

Answer 6

4 parallel sets of reactions, one for each nucleotide

Question 7

What was an initial limitation of Sangers method?

Answer 7

sensitivity, needs lots of DNA

Question 8

For how long was the Sanger method the standard method?

Answer 8

Greater than 20 years

Question 9

What were four improvements of the Sanger method?

Answer 9

1. ddNTPs have different colored fluorescent dyes
2. All 4 ddNTPs can be added into one sequencing reaction
3. Single lane on a gel can be used to sequence the DNA
4. Automated optical read

Question 10

What was an improvement made to increase amplification of gene of interest?

Answer 10

Use of a heat stable polymerase to amplify products

Question 11

what technology was the first capable of taking on the genome of humans?

Answer 11

CE (capillary electrophoresis)

Question 12

What are the two types of primers used for genome sequencing?

Answer 12

1. Universal -forward and reverse

2. Custom-designed “internal” primer

Question 13

Why would a custom-designed primer be needed?

Answer 13

Sequencing technology could only handle 1200 bp sequences. Anything longer than that, you need to break up the sequence into parts. A custom made primer would start a sequence part way through the gene.

Question 14

Define “paired end reads”

Answer 14

short sequences determined from opposite ends of an insert (of known approximate length) using forward and reverse primers

Question 15

what is a sequencing gap?

Answer 15

the last end of sequence that cannot be sequenced due to long length or inability of the primer you have chosen

Question 16

What is a physical gap?

Answer 16

A particular region of the genome not represented in the clone library

Question 17

In the event of a physical gap, how can use another vector to help?

Answer 17

You can prepare a second clone library from the new plasmid, exhibiting stability in the areas that were unstable in the first plasmid

Question 18

What is another name for a probe?

Answer 18

Oligomers

Question 19

How can be oligomers be used to screen a second clone library

Answer 19

They can be used to map the endings of contigs from the first library, demonstrating overlap

Question 20

What are three ways of assembling info from clones into

Answer 20

1. Chromosome walking by hybridization
2. Chromosome walking by PCR
3. Clone fingerprinting