Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

GENE221 > Lecute 18 > Flashcards

Lecute 18 Flashcards

1
Q

What is a BAC?

A

Bacterial artificial chromosome

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are bacterial artificial chromosomes (bacs)

A
  • they are plasmids with features from the F plasmid
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Features of the bacterial artificial chromosome

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is a genomic library ? Definition

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

3 reasons we may want to use a genomic library

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

How to make genomic libraries

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

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

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

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

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

A

Yes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Problem when making a gene library with eulkaryotes

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is the solution to eukaryotes containing introns

A
  • use cDNA
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is cDNA

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

A cDNA library is a collection of…

A

… all expressed RNAs

Mature RNA is isolated from a particular tissue of an organ

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How do u get a cDNA library

A
  • make ur own cDNA library
  • order cDNA clones (availability can be an issue)
  • synthesise cDNA (can be expensive us a large gene)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Two types of libraries

A
  • genomic library - total DNA
  • expression (cDNA) library - all expressed sequences, use expression plasmid vector
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What does sequencing a gene allow us to do?

A
  • 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
17
Q

Process of Sanger sequencing

A
  • 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)
18
Q

When do we use Sanger vs whole genome shotgun sequencing ?

A

Idk figure it out

19
Q

Whole genome shotgun sequencing - i dont understand

A
  • 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)

20
Q

What is haemophilus influenzae

A

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
21
Q

Good things about whole genome shotgun sequencing

A
  • 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
22
Q

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

A

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

23
Q
A

GENE221 (23 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions