molecular biology techniques Flashcards
what is molecular cloning used for
used to isolate a specific sequence of DNA of interest known as an insert
it can be used to sequence a gene, identify mutant genes or express and purify proteins
what are the 3 main stages of molecular cloning
- making recombinant DNA by joining an insert to a vector
- introducing the vector to a host cell
- selecting the host cells that contain rDNA to replicate
what are the most commonly used vector and why are the limitations
plasmids
the size of the insert that can be joined, the number of times it can be copied and how it is applied to the host cell is limited
what are the 4 things all vectors contain
an origin of replication: allows the vector and DNA to be replicated inside the host cell
a selectable marker: to identify cells that have taken up the vector
multiple cloning sites: where insert is cloned
restriction enzymes: to cut DNA at the specific sequence
what are restriction enzymes
enzymes that recognise and bind to specific sequences of DNA called recognition sites and cut them
what are the 4 main types of restriction enzyme
types 1 and 3: cut DNA randomly far away from recognition sites
type 2: cuts DNA at a defined position close to recognition sites
type 4: cuts modified DNA
what is the structure of type 2 restriction enzymes and what do they do
protein homodimers (2 identical proteins joined)
they bind to a non-specific DNA sequence and move to the recognition site where they cut it
they form a palindromic sequence with blunt or sticky overhangs
there is always a 5’ phosphate and 3’ hydroxyl
what are overhanging ends
single-stranded sequences of DNA left after cutting
they can be sticky if cut diagonally or blunt if cut straight
why are sticky ends useful
contain unpaired bases that can bind by complementary base pairing to sequences cut with the same restriction enzyme
what is DNA ligase and what is its role
an enzyme that joins the insertion to the vector by forming phosphodiester bonds between the 5’ phosphate and 3’ hydroxyl group
what are the 2 main problems in molecular cloning and how are they solved
the recognition site could be in the wrong place: polynucleotide kinase adds a phosphate group
DNA present in small amounts so unstable to clone: amplified to PCR
what are nicked ends
the 5’ phosphate group at the ends of the vector can be removed to prevent the ends ligating before the insert has joined
what are the 2 methods of introducing a vector to a host cell
the host cell is shocked with a high-voltage to create pores in the membrane (electroporation) so the vector can enter
the cell can be chemically treated and heat shocked
how are the host cells selected
a marker such as an antibiotic resistance gene is used in the rDNA
the plasmid contains the gene for resistance so any host cells containing the plasmid will survive and can be identified
how can you find the amount of DNA after each PCR cycle
number of PCR cycles to the power of 2
what are the 6 things needed for PCR
a DNA template
DNA polymerase
primers
dNTPs (nucleotides)
a buffer
a thermocycler
what is the process of PCR
- denaturation: heat is applied to break hydrogen bonds and separate DNA strands into ssDNA
- primer annealing: primers bind antiparallel to complementary sequence on ssDNA
- primer extension: DNA polymerase synthesises new strands of DNA at the 3’ end of the primers
what are the features of PCR primers
must be specific to the correct DNA sequence
come in pairs: one binds to the top strand and the other binds to the bottom in the opposite orientation so DNA is amplified between them
must have appropriate melting points to ensure they bind to the right DNA sequence
how is rDNA extracted from bacteria
bacteria is grown in liquid medium and broken open to extract DNA from plasmids
what is the process of gel electrophoresis
- DNA is inserted into wells and an electrical current is applied to the gel
- DNA fragments move towards the positive electrode, smaller fragments move further down
- there is a dye within the gel that sticks to DNA fragments and fluoresces under UV light creating a DNA band
- the band can be compared against fragments of known size
why is DNA sequencing used
to check that no PCR-induced mutations have occurred during DNA cloning
what are modified dNTPs (ddNTPs) in DNA sequncing
nucleotides with a hydrogen at the 3’ end instead of a hydroxyl group so can’t have new nucleotides attached to them
their base is known so we will know which base is at the end of the sequence
what is restriction digest used for
to check that the insert has joined the vector in the correct orientation
how is restriction digest used to analyse DNA
the vector and insert have recognition sites so if the insert is in the wrong orientation the recognition sites won’t line up
this is repeated with many different restriction enzymes
how is PCR used to analyse DNA
the two primers in PCR should face each other and DNA is amplified between them
if the insert isn’t in the right place the primers won’t face each other and a PCR product won’t be made
how can PCR be used in genetic fingerprinting
amplifies the number of repeats, some diseases cause more repeats in DNA which can be identified during PCR
what is the central dogma of cell biology
the theory that DNA is transcribed into RNA which is translated into a protein so gene expression is the production of a functional protein from a gene
how can PCR be used to analyse gene expression
primers can be added to a sample to see which isoforms of mRNA the sample contains
the isoforms can be identified by gel electrophoresis or restriction enzyme digest
how are hybridisation-based techniques used to analyse gene expression
DNA and RNA are complementary so will hybridise
a complementary RNA probe for a specific DNA sequence can be made and is labelled (fluorescent or radioactive)
the probe can be identified to find a specific sequence in a gene
how is northern blot used to analyse gene expression
RNA is separated by size by gel electrophoresis and is transferred to a membrane where a probe is added to tell us about size, isoforms and expression of RNA
how are microarrays used to analyse gene expression
- oglionucleotides (short DNA molecules) are attached to a spot on a chip that all correspond to a different gene
- RNA is fluorescently labelled and made into cDNA which is applied to the chip and allowed to hybridise to the oglionucleotides
this allows us to analyse the expression profile of specific cells
how are reporter genes used to analyse gene expression
reporter genes can be used to identify protein localisation and measure RNA expression by measuring how promoter activity because they are produced when specific proteins are present
how are protein-specific antibodies used to analyse gene expression
- proteins are separated by gel electrophoresis and transferred to a membrane
- the primary antibodies are added and then a secondary antibody that is specific to the primary one is added
- the secondary antibodies are usually labelled so can be identified
this can tell us about protein levels and isoforms
how are fusion proteins used to analyse gene expression
the coding sequence of the protein of interest and fluorescent protein are fused and translated into a single fusion protein
they are used to identify protein isotopes and the relative amount of protein in a sample
how can pull-down assay be used to analyse protein interactions
uses fusion proteins with an affinity for a specific ligand and are purified by another molecule to isolate the protein of interest
this allows us to see what other proteins interact with the isolated protein
how is immunoprecipitation used to analyse protein interactions
uses antibodies that bind to the protein of interest to see what interacts with it
how can yeast two-hybrid be used to analyse protein interactions
- the DNA binding domain of a transcription factor binds to the protein of interest
- for transcription to happen another protein has to bind to the activator domain
- if transcription is stimulated there must be protein interactions
how can chromatin immunoprecipitation (ChIP) be used to analyse protein interactions
- an antibody or fusion protein isolates the protein of interest
- an assay is used to isolate DNA molecules that are associated with the protein
- DNA is cross-linked to the protein and cut up
- the DNA is purified so DNA that hasn’t interacted with the protein is removed
what are the advantages of the DNA polymerase Taq in PCR
good thermostability
rapid extension rate
high efficiency
what are the disadvantages of the DNA polymerase Taq in PCR
no proof-reading ability
low fidelity (accuracy)
forms 3’ adenine overhangs
what happens if the primer annealing temperature is too low or high
if it is too low the primer will bind non-specifically to other sequences
if it is too high the primers won’t bind to DNA and there will be lower product yield
what is the process of RT-PCR
- RNA is converted into cDNA using reverse transcriptase and poly(dT) primers bind to the poly(A) tail of mRNA to remove RNA
- The cDNA is synthesised using the klenow fragment if polymerase I, the hairpin formed by reverse transcriptase acts as a primer and the ssDNA loop is digested by a nuclease
- PCR happens as normal
how does quantitative PCR (qPCR)
SYBR green fluorescent dye or specific fluorescent probes can be used to measure the amount of PCR product
what is cycle threshold and what does it show
Ct (cycle threshold level) is measured when fluorescence exceeds background levels
the difference between Ct values is a relative measure of which sample had the most template at the start
