Lecture 4 Strobel - Sequencing Genomes Flashcards
(28 cards)
Define a genome.
the complete set of genes present in a cell or organism
What is a transcriptome?
The complete set of coding and non-coding RNA transcripts in a cell, population of cells, or organism
What is a proteome?
the complete set of proteins that are expressed by a cell, population of cells, or organism
What was the goal of the Human Genome Project when it was launched in 1990?
they had a goal of determining the complete sequencing of the human genome
What does the term “High-throughput sequencing” refer to?
its a general term that refers to any of several technologies that can sequence DNA in a rapid and cost effective manner.
* eg. Illumina/Solexa
What is the goal of high-throughput DNA sequencing?
to determine the sequence of millions to billions of DNA sequences simultaneously
Illumina sequencing sequences by what?
synthesis
Explain what happens during Illumina sequencing.
- nucleotides are attached to fluorescent dyes (different dyes for each base)
- synthesize a new DNA strand one nucleotide at a time and read the sequence based on fluorescence
What are the advantages of sequencing by synthesis (Illumina sequencing)?
- it can sequence billions of different DNA molecules simultaneously
- there is high accuracy (>99%)
What are the disadvantages of sequencing by synthesis (Illumina sequencing)?
- it requires expensive equipment
- can only be used for short DNA sequences (typically 300 bp)
- can only be directly applied to DNA
What is primer extention?
a technique for in vitro DNA synthesis in which a primer is annealed to DNA and then extended by a DNA polymerase
What is a chain terminator?
a nucleotide that, when incorporated into DNA or RNA, blocks further extension of the nucleic acid chain
What does a reversible chain terminator do?
terminate primer extension reversibly
After a reversible chain terminator is incorporated into DNA, what happens to DNA polymerase?
the DNA polymerase cannot extend the DNA chain further due to a blocking group on the 3’ OH
What happens when you remove the blocking group on the 3’ OH?
removing the blocking group restores the 3’ OH
* once the 3’ OH is restored, DNA synthesis can proceed
Explain what occurs during step 1 of sequencing of a genome using the Illumina platform.
Step 1: Prepare the genomic DNA sample
* Illumina can only accommodate short DNA fragment so you have to break the genomic DNA into small pieces
What does Illumina sequencing require in order for it to work?
adapter sequences that are attached to the ends of the fragmented genomic DNA
Explain what occurs during step 2 of sequencing of a genome using the Illumina platform.
Step 2: attach the DNA to the surface
* the DNA library is denatured (made single stranded) and attached to primers on a glass surface
* the primers are complementary to sequences in the adapters
What is the significance of attaching DNA to a glass surface?
this helps the DNA to spread out different DNA molecules
what occurs during step 3 of sequencing of a genome using the Illumina platform.
Bridge Amplification
* DNA bridges over to another primer on the glass surface
what occurs during step 4 of sequencing of a genome using the Illumina platform.
DNA polymerase synthesizes a complementary DNA strand and the fragments become double-stranded
what occurs during step 5 of sequencing of a genome using the Illumina platform.
the DNA has been duplicated and the process is repeated
What does Illumina sequencing result in?
clusters of DNA molecules that are derived from the original genomic library
What occurs during nanopore DNA sequencing?
- DNA is threaded through a nanopore
- DNA sequence is read as changes in current on either side of the pore
