Lecute 18 Flashcards
What is a BAC?
Bacterial artificial chromosome
What are bacterial artificial chromosomes (bacs)
- they are plasmids with features from the F plasmid
Features of the bacterial artificial chromosome
- Low-copy number origin of replication (1-2 per cell)
- Contain genes to stabilise plasmid
- Antibiotic resistance marker
- Can carry large DNA inserts
What is a genomic library ? Definition
A collection of all the genomic DNA fragments of a given species that have been taken from one organism and inserted into a type of vector for cloning
The aim of this is to have a panel of bacteria containing individual clones that represent all of the DNA in an organisms genome
3 reasons we may want to use a genomic library
To isolate genes for biotechnology
To identify the genes in an organism
To obtain the genome sequence and gene function
How to make genomic libraries
- take the entire genomic DNA (eukaryotic,prokaryotic, viral)
- cut all the DNA using
- RE digest OR
- Shear the DNA (get random cuts)
Then go and clone it
Why we must use the bacterial artificial chromosome vector when cloning the genomic library
Cos a plasmid as 20kb which means you would need so many more compared to bac which is 150kb
What can we use to look for expression on prokaryotic DNA? How?
- procaryotic DNA
- transcribe it into mRNA
- translated into proteins
Therefore genomic library can be used to look for expression of prokaryotic DNA
Remember when making a gene library the eukaryotic DNA has to be spliced to remember the introns before translation
Yes
Problem when making a gene library with eulkaryotes
It contains introns
Introns interrupt genes, exons are the expressed portion
Introns make genes large (hard to get whole gene in a single plasmid clone)
Many recombinant expression systems can’t process introns
Often want to know what is expressed from a gene
What is the solution to eukaryotes containing introns
- use cDNA
What is cDNA
CDNA ( complementary DNA) is a DNA copy synthesised from mRNA using a viral enzyme, reverse transcriptase (RT)
This results in DNA seuquences lacking introns so genes can be translated into proteins in bacteria
Smaller fragments
Restriction enzymes not required
A cDNA library is a collection of…
… all expressed RNAs
Mature RNA is isolated from a particular tissue of an organ
How do u get a cDNA library
- make ur own cDNA library
- order cDNA clones (availability can be an issue)
- synthesise cDNA (can be expensive us a large gene)
Two types of libraries
- genomic library - total DNA
- expression (cDNA) library - all expressed sequences, use expression plasmid vector
What does sequencing a gene allow us to do?
- determine the organisation of the gene
- find out what the gene encodes - the function of the RNA or protein it encodes
- we can compare the sequence with different organisms
Process of Sanger sequencing
- stand separates
- anneal primer
- extend with 4 dideoxy nucleotides, each with a different fluorescent dye (in the video she said when nucleotides are being added they are just the normal ones - the coloured ones are only at the end)
- polymerase extends primer and get range of extended products ended by dye labeled terminators
- seperate on a capillary gel then detect bands with a laser detector (from shortest to longest - getting the sequence)
When do we use Sanger vs whole genome shotgun sequencing ?
Idk figure it out
Whole genome shotgun sequencing - i dont understand
- genome cut into many random fragments and then cloned into a particular vector
- (becuase the vector, when we clone the DNA, we’ve got sequence information we can use as a primer for Sanger sequencing, so they used the edge of the vector to sequence into the DNA)
- sequence from ends of a large number of random clones (sequence each fragment)
- output is a random collection of short sequences, some of them overlapping
- assemble sequences on a computer (overlap sequence reads)
- overlapping reads are assembled into contigs
- contigs are joined together
(By using the edge of the vector sequence, could sequence as far as the sequencing would go and get an output of a heap of random sequences)
What is haemophilus influenzae
Bacteria that causes meningitis and ear infections
- the first bacterial genome to be sequenced (1995)
- 1.8 Mb
- sequenced by TIGR using whole genome shotgun sequencing methods
Good things about whole genome shotgun sequencing
- sequence from ends of a large number of random clones
- assmembe sequences on a computer
- fill in gaps with targeted sequencing later
- it was faster and cheaper then the ordered approach
- aautomoation using Sanger sequencing made this approach excellent at the time
Replication of plasmid DNA, purification and linearisation and m RNA and encapsulation and packing vials
Plasmid DNA containing SARS-CoV-2 spike protein inserted into bacteria
Bacteria replicate in large vats for 2 weeks - trillions of plasmids are produced
Plasmid DNA is purified out of the E.coli
Plasmid DNA linearised using restriction enzymes
Linearised DNA stored in bags and frozen at -80 degrees
Pfizer checks each nag to ensure all the DNA are exact copies
Shipped to massachusertts or Germany
Spike protein DNA is transcribed into mRNA
- this takes place in a 40L vessel (takes 2-4 days)
- each vessel produces 10 million doses of vaccine
- the Andover plant will run approx 4 batches per week
- filtered sterile environment, samples are constantly taken and tested
- mRNA is frozen in bags at -80 degrees and shipped to Michigan
Next step is in the photo below
