Module 1.4: Misc Flashcards
Describe the process of DNA PCR
two strands separated by helicase
annealing of primers to the dissociated DNA strands (temp lowered)
complimentary sequence recreated for both strands by DNA polymerase
cycle is repeated
Describe the central dogma of molecular biology
DNA replicated to make RNA to make proteins
This can be reversed in viruses who are made up of RNA
DNA -transcription–> pre-mRNa –splicing–> mRNA –translation–> proteins –> post-translational modifications
Describe the process of chain termination
Developed by Sanger
most important methods of DNA sequencing
ddNTPs used to terminate DNA elongation because they lack a 3’-OH
These may be radioactively/fluorescently labelled for detection
4 lanes are needed to sequence each of the bases - each set of reactions will have one of the 4 ddNTP base pairs
What are the limitations of chain termination?
the larger the fragment, the less the separation and resolution
max amount of DNA that can be required reliably is 500-1000 bases
can only be used for small scale projects
DNA can be sequence multiple times and compared –> decrease the chances of error
Describe new generation sequencing: Pyrosequencing
- synthesis one base at a time, with real time detection
- a mixture of three enzymes - DNA polymerase, ATP sulfurylase and firefly luciferase - and a nucleotide added to a single stranded DNA. Incorporation detected by light emmited
The process is repeated for each of the 4 nucleotides
Describe new generation sequencing: Illumina genome analysis
DNA molecules and primers ar first attached on a slide or flow cell and amplified with polymerase so that one long strand of >150m single molecules are hypbrised into a double stranded chain
This is denatured and the original template is washed away
single strand then flips over and hybrdises to adjacent primers to form a bridge which is extended by polymerase and double strands form. This is denatured to form two single stranded temptates
this is repeated until multiple bridges are formed and denatured to form single strands
The sequencing primer is then hybridsed to adaptor sequence
Detect fluorescence
HIGH QUALITY BUT SHORT
Describe new generation sequencing: SMRT
DNA synthesised in zero-mode wave-guides (ZMWs)
the sequencing is performed using a polymerase attached to ZMW bottom and fluorescently labelled nucleotides flowing freely in the solution
Only the fluorescence occuring at the bottom of the well is detected.
The fluorescent label is detached from the nucleotide upon its incorporation into the DNA strand, leaving an unomdified DNA strand
LONG READS, GREATER ERRORS
Describe nanopore sequencing and its advantages
THIRD GENERATION
preliminary experimental stage
ssDNA molecules can be electrophorecally driven in a strict linear sequence through a biological pore and can be detected given that the molecules release an ionic current while moving through the pore
The pore can recognise each base
Length is irrelevant, resolution depends on distance between each base
No assembly issues
NO CLONING INVOLVED!!! - will get regions not covered by Sangers
Potentially get complete chromosome sequences
Sequence of both chromosomes - mom and dad
Define microbiota
the qualitative and quantitative information about the different microbes present in a system
If a baby is born via C-section, it will have bacteria found on skin in the gut e.g. s aureus
If a baby is born naturally, it will have bacteria found in the vagina in the gut e.g. lactobacilli
Define microbiome
the functions that these microbiome have
microbiome is host specific and can be changed by diet, pregnancy and surgery
define metegenomics
gain of function or DNA based approach to create gene catalogues used to define the microbiome
Define Metataxonomics
creation of 16S rRNA gene inventories used to define the microbiota
Define metabolomics
catalogue of metabolites in a sample
What are some benefits and risks of microbiota
BENEFITS
- defence - bacterial antagonism
- priming of mucosal immunity
- peristalsis
- metabolism of dietary carcinogens
- synthesis of B and K vitamins
- epithelial nutrients
- conversion of pro-drugs
- utilisation of indigestible carbs
RISKS
- can lead to carcinogenesis
- overgrowth syndromes
- opportunism and translocation
- Essential ingredient for IBD
- implicated in obesity, metabolic syndrome and CRC
What are the ‘omic’ approaches to investigate the gut microbiome
Bottom Up Approach
- metataxonomics = identify what species are there and their abundance
- metagenomics = identify what they do
- This can be used to predict the risk of developing specific diseases and develop drugs to inhibit them
- flow of information: DNA –> RNA –> protein –> metabolite
Top Down Approach
- looking at metabolite pool in specific tissue and samples such as urine or blood to determine the microorganisms and what they do
