Lecute 18

Question 1

What is a BAC?

Answer 1

Bacterial artificial chromosome

Question 2

What are bacterial artificial chromosomes (bacs)

Answer 2

• they are plasmids with features from the F plasmid

Question 3

Features of the bacterial artificial chromosome

Answer 3

1. Low-copy number origin of replication (1-2 per cell)
2. Contain genes to stabilise plasmid
3. Antibiotic resistance marker
4. Can carry large DNA inserts

Question 4

What is a genomic library ? Definition

Answer 4

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

Question 5

3 reasons we may want to use a genomic library

Answer 5

To isolate genes for biotechnology
To identify the genes in an organism
To obtain the genome sequence and gene function

Question 6

How to make genomic libraries

Answer 6

• 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

Question 7

Why we must use the bacterial artificial chromosome vector when cloning the genomic library

Answer 7

Cos a plasmid as 20kb which means you would need so many more compared to bac which is 150kb

Question 8

What can we use to look for expression on prokaryotic DNA? How?

Answer 8

• procaryotic DNA
• transcribe it into mRNA
• translated into proteins

Therefore genomic library can be used to look for expression of prokaryotic DNA

Question 9

Remember when making a gene library the eukaryotic DNA has to be spliced to remember the introns before translation

Answer 9

Yes

Question 10

Problem when making a gene library with eulkaryotes

Answer 10

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

Question 11

What is the solution to eukaryotes containing introns

Answer 11

• use cDNA

Question 12

What is cDNA

Answer 12

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

Question 13

A cDNA library is a collection of…

Answer 13

… all expressed RNAs

Mature RNA is isolated from a particular tissue of an organ

Question 14

How do u get a cDNA library

Answer 14

• make ur own cDNA library
• order cDNA clones (availability can be an issue)
• synthesise cDNA (can be expensive us a large gene)

Question 15

Two types of libraries

Answer 15

• genomic library - total DNA
• expression (cDNA) library - all expressed sequences, use expression plasmid vector

Question 16

What does sequencing a gene allow us to do?

Answer 16

• 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

Question 17

Process of Sanger sequencing

Answer 17

• 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)

Question 18

When do we use Sanger vs whole genome shotgun sequencing ?

Answer 18

Idk figure it out

Question 19

Whole genome shotgun sequencing - i dont understand

Answer 19

• 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)

Question 20

What is haemophilus influenzae

Answer 20

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

Question 21

Good things about whole genome shotgun sequencing

Answer 21

• 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

Question 22

Replication of plasmid DNA, purification and linearisation and m RNA and encapsulation and packing vials

Answer 22

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

Question 23