molecular biology techniques

Question 1

what is molecular cloning used for

Answer 1

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

Question 2

what are the 3 main stages of molecular cloning

Answer 2

1. making recombinant DNA by joining an insert to a vector
2. introducing the vector to a host cell
3. selecting the host cells that contain rDNA to replicate

Question 3

what are the most commonly used vector and why are the limitations

Answer 3

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

Question 4

what are the 4 things all vectors contain

Answer 4

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

Question 5

what are restriction enzymes

Answer 5

enzymes that recognise and bind to specific sequences of DNA called recognition sites and cut them

Question 6

what are the 4 main types of restriction enzyme

Answer 6

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

Question 7

what is the structure of type 2 restriction enzymes and what do they do

Answer 7

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

Question 8

what are overhanging ends

Answer 8

single-stranded sequences of DNA left after cutting
they can be sticky if cut diagonally or blunt if cut straight

Question 9

why are sticky ends useful

Answer 9

contain unpaired bases that can bind by complementary base pairing to sequences cut with the same restriction enzyme

Question 10

what is DNA ligase and what is its role

Answer 10

an enzyme that joins the insertion to the vector by forming phosphodiester bonds between the 5’ phosphate and 3’ hydroxyl group

Question 11

what are the 2 main problems in molecular cloning and how are they solved

Answer 11

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

Question 12

what are nicked ends

Answer 12

the 5’ phosphate group at the ends of the vector can be removed to prevent the ends ligating before the insert has joined

Question 13

what are the 2 methods of introducing a vector to a host cell

Answer 13

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

Question 14

how are the host cells selected

Answer 14

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

Question 15

how can you find the amount of DNA after each PCR cycle

Answer 15

number of PCR cycles to the power of 2

Question 16

what are the 6 things needed for PCR

Answer 16

a DNA template
DNA polymerase
primers
dNTPs (nucleotides)
a buffer
a thermocycler

Question 17

what is the process of PCR

Answer 17

1. denaturation: heat is applied to break hydrogen bonds and separate DNA strands into ssDNA
2. primer annealing: primers bind antiparallel to complementary sequence on ssDNA
3. primer extension: DNA polymerase synthesises new strands of DNA at the 3’ end of the primers

Question 18

what are the features of PCR primers

Answer 18

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

Question 19

how is rDNA extracted from bacteria

Answer 19

bacteria is grown in liquid medium and broken open to extract DNA from plasmids

Question 20

what is the process of gel electrophoresis

Answer 20

1. DNA is inserted into wells and an electrical current is applied to the gel
2. DNA fragments move towards the positive electrode, smaller fragments move further down
3. there is a dye within the gel that sticks to DNA fragments and fluoresces under UV light creating a DNA band
4. the band can be compared against fragments of known size

Question 21

why is DNA sequencing used

Answer 21

to check that no PCR-induced mutations have occurred during DNA cloning

Question 22

what are modified dNTPs (ddNTPs) in DNA sequncing

Answer 22

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

Question 23

what is restriction digest used for

Answer 23

to check that the insert has joined the vector in the correct orientation

Question 24

how is restriction digest used to analyse DNA

Answer 24

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

Question 25

how is PCR used to analyse DNA

Answer 25

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

Question 26

how can PCR be used in genetic fingerprinting

Answer 26

amplifies the number of repeats, some diseases cause more repeats in DNA which can be identified during PCR

Question 27

what is the central dogma of cell biology

Answer 27

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

Question 28

how can PCR be used to analyse gene expression

Answer 28

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

Question 29

how are hybridisation-based techniques used to analyse gene expression

Answer 29

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

Question 30

how is northern blot used to analyse gene expression

Answer 30

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

Question 31

how are microarrays used to analyse gene expression

Answer 31

1. oglionucleotides (short DNA molecules) are attached to a spot on a chip that all correspond to a different gene 2. 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

Question 32

how are reporter genes used to analyse gene expression

Answer 32

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

Question 33

how are protein-specific antibodies used to analyse gene expression

Answer 33

1. proteins are separated by gel electrophoresis and transferred to a membrane 2. the primary antibodies are added and then a secondary antibody that is specific to the primary one is added 3. the secondary antibodies are usually labelled so can be identified this can tell us about protein levels and isoforms

Question 34

how are fusion proteins used to analyse gene expression

Answer 34

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

Question 35

how can pull-down assay be used to analyse protein interactions

Answer 35

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

Question 36

how is immunoprecipitation used to analyse protein interactions

Answer 36

uses antibodies that bind to the protein of interest to see what interacts with it

Question 37

how can yeast two-hybrid be used to analyse protein interactions

Answer 37

1. the DNA binding domain of a transcription factor binds to the protein of interest 2. for transcription to happen another protein has to bind to the activator domain 3. if transcription is stimulated there must be protein interactions

Question 38

how can chromatin immunoprecipitation (ChIP) be used to analyse protein interactions

Answer 38

1. an antibody or fusion protein isolates the protein of interest 2. an assay is used to isolate DNA molecules that are associated with the protein 3. DNA is cross-linked to the protein and cut up 4. the DNA is purified so DNA that hasn't interacted with the protein is removed

Question 39

what are the advantages of the DNA polymerase Taq in PCR

Answer 39

good thermostability rapid extension rate high efficiency

Question 40

what are the disadvantages of the DNA polymerase Taq in PCR

Answer 40

no proof-reading ability low fidelity (accuracy) forms 3' adenine overhangs

Question 41

what happens if the primer annealing temperature is too low or high

Answer 41

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

Question 42

what is the process of RT-PCR

Answer 42

1. RNA is converted into cDNA using reverse transcriptase and poly(dT) primers bind to the poly(A) tail of mRNA to remove RNA 2. 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 3. PCR happens as normal

Question 43

how does quantitative PCR (qPCR)

Answer 43

SYBR green fluorescent dye or specific fluorescent probes can be used to measure the amount of PCR product

Question 44

what is cycle threshold and what does it show

Answer 44

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