Flashcards in Lecture 6: Sanger and sequencing Deck (20)
Define tandem repeats
Repeats in DNA that are directly adjacent to each other
Define dispersed repeats
repeats found in varying regions of the genome
Walter Fiers was able to do what in 1972?`
He sequenced the first protein coding gene, from RNA bacteriophage MS2
What was first genome to be sequenced?
RNA bacteriophage MS2
Describe Sanger chain termination method (3 points)
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
How many parallel sets of reactions take place in Sanger chain termination method
4 parallel sets of reactions, one for each nucleotide
What was an initial limitation of Sangers method?
sensitivity, needs lots of DNA
For how long was the Sanger method the standard method?
Greater than 20 years
What were four improvements of the Sanger method?
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
What was an improvement made to increase amplification of gene of interest?
Use of a heat stable polymerase to amplify products
what technology was the first capable of taking on the genome of humans?
CE (capillary electrophoresis)
What are the two types of primers used for genome sequencing?
1. Universal -forward and reverse
2. Custom-designed "internal" primer
Why would a custom-designed primer be needed?
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.
Define "paired end reads"
short sequences determined from opposite ends of an insert (of known approximate length) using forward and reverse primers
what is a sequencing gap?
the last end of sequence that cannot be sequenced due to long length or inability of the primer you have chosen
What is a physical gap?
A particular region of the genome not represented in the clone library
In the event of a physical gap, how can use another vector to help?
You can prepare a second clone library from the new plasmid, exhibiting stability in the areas that were unstable in the first plasmid
What is another name for a probe?
How can be oligomers be used to screen a second clone library
They can be used to map the endings of contigs from the first library, demonstrating overlap