Week 6 Flashcards
What is a genome
The complete genetic material present in a cell
What is the transcriptome
Sum of the total mRNA expressed from the genes
Proteome
The entire complement of protein produced and expressed by a cell, tissue or organism
functional assays
systemic experiments used to explain the function of a protein in a cellular pathway and gene activity
include - flow cytometry,
what are the two DNA cloning methods
restriction enzymes
cloning vectors
restriction enzymes
used to cleave DNA at specific sites
cloning vectors
bacterial plasmids or viruses
to carry inserted foreign fragments
what is DNA cloning technique
joining together sequences from different sources
also called recombinant DNA
what is genetic engineering
changing genetic content
additions and deletions will be inherited
includes artificial changes in the DNA content
becomes part of the genome
creates a transgenic organism
genetic engineering process
- vector and DNA fragment
- forms recombinant DNA
- recombinant DNA replicates within host cells
- isolation, sequencing and manipulation of purified DNA fragment
what are nucleases
- hydrolyse an ester within a phosphodiester bond
- can be dna or rna specific
needed for specific reassembly of dna fragments
what are the nucleases
endonucleases
restriction endonucleases - type II endonucleases
isoschizomers
neoschizomers
what are endonucleases
cleave WITHIN a nucleic acid chain - dna or rna
what are exonucleases
cleave from either END of dna or rna
what are restriction endonucleases
- are bacterial enzymes
- needed for production of recombinant dna
- can cleave double stranded dna at specific sites
example - type II endonucleases
what are bacterial enzymes
enzymes found naturally in bacteria
what are type II endonucleases
most commonly used restriction endonuclease
same recognition and cleavage site
different from type I as those sites are far from each other
specificity may vary - some might allow similar sequences
how can type II restriction endonucleases be cut
can be staggered with sticky ends
- single stranded complementary overlap
blunt double stranded cut
- no overlap
- can still be useful in labs
what are isoschizomers
pairs of two different enzymes
both specific to the same recognition site
cut in the same way
what are neoschizomers
- different enzymes with the same recognition site
- cut differently can create different sticky ends
what is a modification enzyme
produced by bacteria for each restriction enzyme with the same recognition sequence - usually a DNA methyltransferase
as a methylated recognition sequence cannot be cleaved by the restriction enzyme
a bacteriums own genome is protected by modification
what is a vector
cloning requires a specifically engineered vector
often as a plasmid
can be used to propagate an incorporated dna sequence in the host
what are some features of vectors
ORI, selective marker, engineered multiple cloning site, dna ligase
what is an ORI
origin of replication
dna sequence that signals the start
what is a selective marker
identifies transformants to allow for growth
and eliminates non-transformants
what are transformants
cells with foreign genetic material
engineered multiple cloning site
a synthetically generated sequence of DNA
contains multiple restriction sites
series of tandem (two or more) restriction endonuclease sites
usd in cloning vectors to create recombinant molecules
different enzymes can be chosen to create specific sticky ends
what is dna ligase
used for dna fragments with sticky or blunt ends
How is bacterial transformation done in the lab
Done by calcium chloride
And heat shock
Not effective so selective marker is used to collect the recombinant dna that have acquired antibiotic resistance
Why is bacterial amplification and purification done
To clear negative bacteria
And select the positive bacteria that contain the plasmid
How is bacteria containing the plasmid amplified
Colonies are selected and grown in liquid broth containing the antibiotic
Negative bacteria dies out and positive bacteria survive and give rise to more colonies
How is plasmid dna isolated from the bacteria
Alpine lysis based spin colomn
How do false positives of plasmids occur
Plasmid recircularises without a cloned fragment
What is blue white selection
Allows bacteria with a vector and insert to be identified
How does blue white screening work
The multiple cloning site is in the LacZ operant
LacZ produced beta galactosidase which turns blue when X-gal is added
So plasmids that contain the dna fragment interrupt beta galactocidase activity - and appear white
The white colonies are selected
What is a dna library
Collection of dna molecules that are cloned into a vector
Effectively represent all dna sequences in the genome
Pros/cons of dna library
Useful for representing the genome of simple organisms
cDNA is more useful for higher eukaryotes
What is cDNA
Synthetic ‘copy’ DNA
That has been transcribed from mRNA
- only has exons
How is cDNA made
- copies of mRNA are collected and synthesised into plasmid vectors
MRNA is easily collected by a primer for the polyA tail
Enzyme reverse transcriptase is used to synthesise a complementary DNA to each mRNA - makes a single stranded cDNA
Which is then converted into a double stranded molecule
How is cDNA inserted into a vector
Sticky end or linked is needed
Dna pol synthesises a second strand
Fragments are protected by methylation at EcoRi sites
Synthetic linkers with EcoRI restriction site is added to double strand cDNA
What are the limitations of cDNA
Needs to be transformed (put into) E.coli
Only represents expressed DNA - exons
Silent genes not represented
Over expressed genes will be overrepresneted
what is screening the cDNA library
required to identify clones with the interested gene
what is hybridisation
ability of complementary dna or rna molecule
to associate with each other by base pairing
can be used to identify specific nucleic acids - when it is of a labelled oligionucleotide
what is a probe
a radioactive nucleic acid - dna or rna
used to identify a complementary fragment
and locate the DNA sequence
signal appears over plasmid dna that is complementary to the probe
