Comprehensive lecture notes for the Bacterial Genetics and Genomics module covered in MCB3026F. These notes cover all content taught in lectures as well as additional materials (powerpoints, textbooks) required to succeed. These notes were created by a student who achieved a distinction in this cou...
Lecture 1: A pre-NGS example: Chromosomal libraries
- A common way used to study gene function before next generation sequencing
- Example: streptomycin resistance in mycobacteria
Streptomycin resistance
- Streptomycin is an antibiotic effective against mycobacterium tuberculosis
- Mode of action inhibits protein synthesis
- Mechanism of resistance: modification of targets such as mutations in S12 ribosomal protein or 16S rRNA which reduces
the affinity of these proteins for streptomycin causing the bacteria to become resistant and efflux meaning that the bacteria
have an efflux pump which is able to pump the antibiotic out of the cell to the antibiotic cannot reach its targets in the cell
Mycobacterium fortuitum FC1 strain
- Mycobacterium fortuitum is a closely related cousin to mycobacterium tuberculosis (both from the mycobacterium genus)
- Is an opportunistic pathogen and can cause infections in various tissues including skin, bone and lung
- A resistant strain was isolated and had a minimum inhibitory concentration of 50 micrograms per ml (the lowest
concentration of an antibiotic that can inhibit or kill the bacteria) which is higher than normally observed
Generation of chromosomal library
- Generated a chromosomal library to help identify the gene responsible for the resistance
- To do this: needed to extract the chromosomal DNA from the strands
- Once extracted, DNA is broken down into smaller pieces
- Have fragments of the genome of different sizes and each fragment can either have a single gene or part of a gene or
multiple genes
- Each fragment is linked or attached into a carrier piece of DNA called a vector or plasmid
- Plasmid carries the fragment
- Each plasmid carrying a fragment would be transformed into a different cell or bacteria
- Colonies on a plate represents the library where each colony contains a length of DNA from the original strain
- This library would then influence the bacteria that is carrying each of these plasmids
- If the gene responsible for streptomycin resistance is present on the plasmid, then it will make the bacteria carrying that
gene become resistant to streptomycin
-
Used this property to find the gene responsible for the resistance
- Took the clones in the library and selected them by placing them on media containing
streptomycin at a concentration that would normally be lethal for these bacteria
- Only the bacteria containing the gene for resistance would grow
- Selected for these colonies and separated the plasmids by extracting them out of those
bacterial cells
- Have a plasmid containing a fragment with several genes that could be responsible for the resistance
- To find which gene is responsible exactly, used sequencing
- Used sanger sequencing to find the sequence of the bases in the fragment
- Pac5 and 6 plasmids both contained a bigger fragment and the common piece of DNA was a 2.5 kilobase piece and this
suggested that the resistance gene is found within this 2.5 kb region
- Can predict the genes that are present and can predict where the different genes
started and ended
- orfA and orfE are both partly cut off so neither of these genes are responsible for the
resistance leaving 3 genes that could be responsible, orfB, orfC and orfD
- Researches cut out a piece so that the fragment only completely contained orfC
Analysis of cloned DNA
- Took the 2 fragments, once containing orfB, C and D and the 1 kb fragment containing
only orfC and cloned each into a new plasmid
, - The plasmid containing the 2.5 kb fragment is called pSAN19 and the plasmid containing the 1 kb fragment is called
pSAN26
- They introduce these plasmids into new bacteria and select on streptomycin and measure the level of resistance to
streptomycin in each case
- Results show the level of resistance seen
- At the M. smegmatis results, for pSUM36 (the plasmid without any fragment) is only able to grow at a concentration of ml
of streptomycin
- When the pSAN19 plasmid is introduced, able to grow at a concentration of 32 mg per ml so it is resistant to streptomycin
again
- When pSAN26 is introduced, has a level of resistance at 32 mg per ml
Lecture 2: Sanger sequencing
- Sanger sequencing was developed by Frederick Sanger in 1977
- Chain-terminating dideoxynucleotides
Sequencing of the cloned DNA
- The method relies on the presence of dideoxynucleotides which function to
terminate the chain during DNA synthesis
- Normally during DNA synthesis, have incoming base which bonds to
template strand by a phosphodiester linkage (P group attacked by the
hydroxyl at the 3’ position of the previous base)
- The dideoxynucleotides are modified and carry only a hydrogen instead of a
hydroxyl at the 3’ position
- The hydrogen cannot link to the phosphate to form the phosphodiester
linkage and the chain cannot be elongated
Sanger Sequencing process
- Isolated DNA is transferred to a plate where the sequencing reaction will take place
- Free DNA bases are added, DNA polymerase enzyme, DNA primers and modified DNA bases labelled with colored
fluorescent tags called terminator bases
- Everything is heated to 96 degrees to start the reaction which separates the DNA into separate single strands
- Temperature lowered to 50 degrees
- Enables DNA primers to bind to the plasmid DNA
- Temperature is increased to 60 degrees
- DNA polymerase binds to the primer DNA
- The enzyme makes new DNA strand by adding unlabeled DNA bases to the target DNA
- Adds bases until a terminator base is added (have been chemically altered so no new bases can be added to the new strand
of DNA) and DNA polymerase falls away
- Reaction is heated to 96 degrees again to separate the new strand from the original strand
- Heating and cooling process is repeated over and over again to produce lots of fragments of different lengths
- To read the sequence the various fragments are separated by length using a process called electrophoresis
- A capillary tube is lowered into each well of the plate and an electrical charge is applied causing the negatively charged
DNA molecules to move through the capillary tube
- Fragments become arranged by size from the shortest to the longest
- A laser makes the terminator bases light up and the colour is detected by a camera and recorded
- Each terminator base is labelled with a different colour (e.g. C is red)
- The sequencing machine records the colour of the terminator bases as a series of colored blocks representing the
terminator base at the end of each fragment of DNA
- Convert colors to letters to get the sequence of the piece of DNA
Limitations of Sanger sequencing
- Sanger methods can only sequence between 300 to 1000 bp
- The quality not very good in the first 15 to 40 bp (primer binds)
- Sequence quality degrades after 700 to 900 bp
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