Module 7 Flashcards
What is heterologous expression?
Expressing a gene in a different host
What is genetic engineering
Using in vitro techniques to alter genes in the lab
What is DNA amplification
DNA replication in a test tube
How to the cycle numbers grow in PCR ?
Each future cycle doubles the number of copies.
What are the PCR steps?
I. Denature DNA
2, annealing → oligonucleotides added
3. Add DNA polymerase
4. Heat and cool
What is reverse transcription PCR
Makes DNA copies from mRNA template
What does gel electrophoresis do?
Separates DNA molecules based on size and charge
What is DNA’s charge?
Negative
What is nuclei acid probing
Used to detect a certain nucleic acid sequence
How is the probe usually labelled or tagged?
With fluorescence
What is a probe?
Probes are complementary to the gene of interest
What is a southern blot
DNA is in the gol and probe is RNA or DNA
What is a northern blot
RNA is in the gel and the probe is DNA or RNA
What is fluorescent in situ hybridization
Uses a fluorescent probe attached to oligonucleotide to target specific nucleic acid sequences in cells
What are the enzymes used for cloning?
Restriction endonucleases
DNA ligase
Reverse transcription
DNA polymerase
What are restriction endonucleases
Enzymes that recognize specific DNA sequences and cut them
What are restriction endonucleases essential for?
In vitro DNA manipulation and gene cloning
What is methylation?
Coding on cells to prevent cutting by restriction enzymes (defense)
Are restriction endonucleases rare in eukaryotes
Yes
What do type 2 restriction endonucleases do?
Cleave DNA within recognition sequence: most useful for specific DNA manipulation
What recognizes palindromes
Type 2 restriction endonucleases
What is also known as an overhang cut
Sticky end cut
What is blunt end cutting
Both strands are cut at the same position
When are sticky ends produced?
When restriction enzymes make staggered cuts
What can molecules with complimentary sticky ends easily do?
Anneal or form hydrogen bonds between complimentary bases
What is ecoR1
A type 2 restriction enzyme that is isolated from strains of E coli
What is each restriction enzyme paired with?
A corresponding modification enzyme that shares the same recognition sequence
How can a gene to be cloned be amplified by? (3)
Polymerase chain reaction
Synthesized by reverse transcriptase
Synthetic DNA made in vitro
What are used as cloning vectors
Plasmids
What are the 3 main steps of gene clothing?
Isolation and fragmentation of source DNA
Insertion of DNA fragment into cloning vector
Transformation of cloned DNA into host organism
What is blue / white screening
Bacteria with a cloning vector inside that may or may not have recombinant DNA
What do blue colonies not have
Plasma vectors with foreign DNA inserted
What do white colonies have
The recombinant DNA
What gene does blue/ white screening rely on?
LacZ gene
Where does the lacZ gene often fuse?
To a portion of the plasmid known as the multiple cloning site
When X gal is cleaved by b-galactosidase, what happens?
It turns blue
Why does a colony stay white?
Because it contains the gene or interest and lacZ gene will be disrupted and b-galactosidase cannot be produced
What is on the plasmid vector
LacZ gene and a multiple cloning site
What are DNA cassettes / cartridges
Synthetic fragments that can make more then a few base pair changes or replace sections of a gene via cassette mutagenesis
what are the properties of an open reading frame (4)
ribosome binding site seuqunce before the start codon
start codon
coding sequence
stop codon
what are the enzymes in DNA cloning
DNA polymerase
Restriction endonuclease
DNA ligase
Reverse transcriptase
whats the purpose of DNA polymerase in cloning
it synthesizes the DNA strand
what is the purpose of restriction endonuclease in DNA cloning
they recognize specific DNA sequences and cut DNA
what is the purpose of DNA ligase in cloning
going two strands of DNA
what is the purpose of reverse transcriptase
converts RNA into DNA
what do expression vectors have that coning vectors don’t
inducible promoter
tag for purification
what are expression vectors for?
they are designed to facilitate gene expression and protein production
what is an inducible promotor?
a DNA sequence that drives the expression of the gene - it controls initiation of transcription by RNA polymerase
what is a cloning vector?
DNA molecule used to carry and replicate foreign DNA fragments in a host organism
what is the best generic method to diagnose an RNA virus
RT-PCR
why is the RT-PCR the best genetic method to diagnose an RNA virus
the reverse transcriptase allows the synthesis of cDNA from RNA
what is genomics
the study of the entire genome (all genes and genetic material)
what us transcriptomics
the study of a transcriptome, which is the complete set of RNA molecules (mainly mRNA)
what is proteomics
the study of the proteome, which is the entire set of proteins expressed by the genome
what is metabolomics?
the study if a metabolome, which is the small molecule metabolites (sugars, lipids, amino acids)
what does transcriptomics focus on understanding
gene expression patterns
what are microarrays
they use pre designed probes to measure the expression levels of thousands of genes at once
what is western blotting used for
measuring protein levels
why are primers required for polymerase chain reactions
to provide a starting point for DNA polymerase
what is a thermocycler
a machine used for PCR reactions
what is the structure of a nucleic acid probe
ss DNA fragments complementary to the sequence of interest
cutting DNA with EcoR1 will produce
5’ overhangs
