Module 7: Microbial Genomics (DNA Sequencing + Analysis) Flashcards
Genome
An organism’s complete set of DNA including ALL of its genes
Genetics
The study of individual genes and their functions
Genomics
The determination and study of complete genome sequences
–> An interdisciplinary field investigating many aspects of genomes
How are genetics and genomics different?
Genetics deals with individual genes whereas genomics deals with the genome as a whole
Genomics is an _________ field that investigates what aspects of genomes? (5)
Interdisciplinary
Investigates Genome…
1) Structure
2) Function
3) Evolution
4) Mapping
5) Editing
Genomic sequences allows us to link genetic characteristics of microbes to their what? (2)
1) Physiological properties
2) Ecological Role
The study of genomes allows us to: (3)
(in an overall sense)
1) Generate hypotheses
2) Investigate Differences (between genomes)
3) Examine evolutionary relationships
Genomic analysis allows for the examination of evolutionary relationships of microbes: this then facillitates the analysis of
medical and epidemiological analysis of infectious disease
The establishment of genomics and molecular biology was made possibly by: (2)
1) the advent of recombinant DNA techniques
2) Development of DNA sequencing methods
What were the first DNA sequencing methods?
Two different methods arose at roughly the same time:
1) Fred Sanger’s Enzymatic Method (Sanger Sequencing)
2) Walter Gilbert + Alan Maxam’s Chemical Degradation Method
DNA Sequencing
The process of determining the nucleic acid sequence of DNA
Most sequencing methods to this day are variations of what?
Variations of the Sanger Sequencing Method
What is Sanger Sequencing also known as?
Sanger Sequencing =
“Dideoxy Sequencing” + “Chain-Termination Method”
Sanger sequencing takes advantage of the ______________ nature of DNA polymerase
What does this mean?
Takes advantage of the PRIMER-SPECIFIC nature of DNA polymerase
== Sequencing methodology is based upon the principle that DNA polymerase requires a free 3’ OH end (typically from a primer) to begin DNA synthesis
What are the 3 main steps of Sanger sequencing?
1) DNA cloning
2) DNA synthesis
3) Gel Electrophoresis
What does DNA polymerase do in DNA synthesis?
Adds nucleotides together via formation of phosphodiester bonds between free 3’ OH and 5’ PO4 ends
dNTPs
deoxyribonucleotide triphosphate –> The building blocks of DNA that get connected together by DNA polymerase
–> Consists of a 5 carbon ribose sugar, a 3’ Carbon hydroxyl group, a 5’ carbon triphosphate, and a 1’ carbon nitrogenous base
ddNTPs
Dideoxyribonucleotide triphosphate
–> Like dNTPs BUT lack a 3’ OH grp and instead has just a hydrogen on the 3’ carbon
What is at the 5’ and 3’ ends of ddNTPs and why is this significant for sanger sequencing?
5’ End = Free phopshate grp
3’ End = Hydrogen atom (no OH)
–> This means that it can ADD to an elongating chain but nothing can be added to IT!
== Important for chain termination in sanger sequencing!
When a ddNTP is incorporated into a growing strand…
Why does this happen?
DNA synthesis stops!
–> Because the added ddNTP has no free 3’OH grp for DNA polymerase to catalyze formation of a new phosphodiester bond with another nucleotide
Why are ddNTPs called “dideoxy” ?
Because they are “deoxygenated” on two carbons (3’ and 2’)
DNA = “deoxy” because it’s “deoxygenated” on one carbon (2’)
Explain phosphodiester bond formation
The free 3’ OH grp of the last added nucleotide to a growing strand reacts with the 5’ PO4 end of a new nucleotide
–> In doing so, water is released, the phosphodiester bond forms and the bond from the first phosphorus to the other 2 phosphate groups is cleaved and pyrophosphate (PP) is released
In Sanger sequencing, synthesis of new DNA strand is purposefully _________________ by doing what?
DNA strand synthesis is purposefully TRUNCATED / terminated
–> Occurs through incorporation of ddNTP into the growing strand = building block with NO 3’OH
What cannot form with a ddNTP?
A phosphodiester bond at its 3’ end
(Lacks OH grp needed to produce this bond at this carbon)