DNA manipulation Flashcards
role of dna polymerase
- synthesises DNA in the 5’ to 3’ direction
- works with other enzymes to add free nucleotides
- require a template and a primer
Reverse transcription process
- mRNA is isolated from cell where gene is active
- poly-A tail added to provide anchor for primer to attach at 3’ end
- primer binds to poly-A tail
- reverse transcriptase uses mRNA as a template to build single stranded DNA with complementary base sequence
- polymerase builds 2nd strand
- results in complementary DNA (cDNA)
when would reverse transcription be useful?
- when specific genes/small sections of DNA are needed
Restriction enzymes:
- come from bacteria (found in prokaryotic cells)
- cleaves phosphodiester bond between deoxyribose sugar of one nucleotide and the phosphate group of the next.
- have a specific recognition site (due to tertiary structure)
- called endonucleases
Recognition site- how long
- 4-6/8 pairs long
- palindrome (read the same way in both directions (5’ to 3’ and 3’ to 5’)
- length determines how often an endonuclease is likely to cut a DNA sequence, or the average distance between cuts
Blunt ends vs sticky ends
Blunt ends produced when strands cut directly opposite each other and leave no overhanging ends.
Sticky ends are overhanging/ cuts not made directly opposite each other, resulting in overhanging ends.
What is ligase
- catalyses joining of pieces of double stranded dna at their sugar phosphate backbone in ligation
- forms phosphodiester (covalent bonds)
- lack of specificity (no specific site) so they can glue everything
- can produce 1 longer piece or circular molecule if fragments have sticky ends at both ends
disadvantage of blunt ends over specific ends
- blunt ends are non specific to sequences in target DNA fragment, not ideal for purpose of ligating specific fragments of DNA
- any blunt ends can ligate
- less control over ortientation of dna insert
- sticky ends have complementary overhangs, ensure specific base pairing
what is gene cloning?
- particular gene is located and copied to produce mutliple copies
- putting gene somewhere where it’s useful in large quantities
number of recognition sites: circular dna vs linear dna
circular: no of fragments= no. of recognition sites (as circular DNA opens up)
linear: no. of fragments = no. of recognition sites +1
Plasmid: what is it, features
- small, circular, double-stranded DNA
-self-replicating as they have origin of replication (ORI) (DNA base sequence where DNA replication of plasmid begins) - replicate independently of bacterial chromosome
Recombinant plasmids: what are they, features (6)
- contain foreign DNA
- ORI- recognised by DNA replication proteins to initiate DNA synthesis
- Antibiotic resistance gene (e.g ampicillin)
- Multiple cloning site (msc) (or polylinker region)- artificial site with multiple unique endonuclease cut sites, allow foreign DNA to easily be inserted
- promoter region- role in when and where gene of interest is expressed
- selectable marker- gene, helps in selection of transformants
- screening marker- gene, confirms plasmid is recombinant (e.g. lacZ) (non antibiotic marker)
What are vectors?
DNA molecules (often plasmids or a virus) that is used as a vehicle to transfer genetic material into other cells
What is bacterial transformation?
bacterial cells take up foreign DNA
bacterial transformation techniques
electroporation:
- cells placed in electric field that shocks them, creates holes in their membrane, allow for plasmid entry
heat shock:
- put in ice-cold salt solution, transferred to 42 degrees for less than a minute, increases fluidity of membrane
what are recombinant proteins?
proteins expressed by recombinant DNA
advantages of producing proteins through expression of cloned genes, instead of other biological sources
- constant and reliable supply
- high level of purity
- larger quantities
- more appealable to patient than using animals
- reduced chance of side effect
- consistent quality
natural insulin pathway and function
- pre-proinsulin
- pro insulin
- insulin
- allows glucose to enter cells, lowering blood glucose
Why is a reporter gene added to recombinant plasmids?
- to form a fusion protein and prevent degradation
- InsA is small and makes a small polypeptide
- susceptible to degradation
- not natural habitat (in our bodies, polypeptides join quickly (more stable and strong)
Purpose of PCR? (polymerase chain reaction)
- amplify amount of target DNA
- each cycle doubles amount (exponential growth)
- valuable when only small sample of DNA available
Role of primers in PCR and what they are
- short single stranded DNA? molecules
- made in a laboratory to have nucleotides complementary to ends of DNA sequence
- ensure DNA replication initiated at required points/ define the region to be amplified
3 step process of PCR:
- Denaturing (95 degrees)
heating separates DNA strands by breaking hydrogen bonds between complementary base pairs - Annealing (55 degrees)
Cooling allows forward and reverse primers bind to 3’ ends of template strand, through forming hydrogen bonds between complementary base pairing - Extention / elongation (72 degrees)
Optimum temp for Taq polymerase, binds to primer and adds free nucleotides through complementary base pairing to builds new strand
process repeated to obtain suffient amount of DNA
Taq polymerase (PCR) origin/ characteristics
- extracted from thermophillic bacteria (lives in hot springs_
- heat resistant properties allow it to not denature at high temperature used to break hydrogen bonds between DNA strands and it’s optimum temp is 72 degrees
PCR components (6)
Template DNA (DNA to be amplified)- don’t just write template DNA though if you can more specific (e.g. insulin gene)
specific forward and reverse Primers: bind to DNA, specify start and finish of DNA sequence to be amplified
Taq polymerase: builds DNA strand by adding nucleotides
Free nucleotides
Buffer: mimic cell conditions, maintains correct pH and provides any required salts
PCR tube
Process to create Insulin first half: (lac Z)
- Isolate lacZ gene
LacZ gene is cut using specific restriction enzymes that create sticky ends - Plasmid vector is cut with same restriction enzyme.
- Plasmid and lacZ gene mixed with ligase (disrupts tetR gene)
- Bacterial transformation (using heat shock or electroporation), bacteria mixed with plasmids (w and w/o lacZ)
bacteria with plasmid: transformed
bacteria without plasmid: untransformed - bacteria spread on nutrient agar plate with antibiotic (amp) to test for transformed bacteria (only tranformed bacteria survive as ampR gene is intact)
- bacteria placed on another nutrient agar plate (tet) to test for recombinant plasmids (bacteria with recombinant plasmids die)
Process to create Insulin second half:
- Gene of interest, InsA, cut using specific restriction enzyme to create sticky ends. Cut plasmid and mix with ligase. (disrupts lacZ)
- Bacteria spread on nutrient agar with X-gal. Recombinant plasmid with insulin will produce white colonies (as no B-gal can be produced). Transformed bacteria with non-recombinant plasmids will turn blue.
- Transformed bacteria produce insulin A-chain peptide which is extraced and purified. Protein produced is fusion protein (B-gal insulin peptide) to prevent insulin A from being degraded by enzymes
- Process repeated for B-chain, purified where B-gal section is removed, A and B chain mixed together to become active insulin
What is gel electrophoresis? When is it used?
- separation of nucleic acids (DNA or RNA) based on fragment size (length in base pairs)
- commonly used after restriction enzymes cut DNA sample, or PCR amplifies sequence
process of gel electrophoresis?
- DNA sample placed in well at one end of agarose gel (sponge like, filled with pores)
- agarose gel immersed in buffer solution (ionic dissolved salt)
- electric field applied to gel with positive pole (anode) at far end and negative pole (cathode) at origin end.
- DNA is negative (due to phosphate group) so it’s attracted to positive end
- smaller fragments move faster
DNA ladder in gel electrophoresis
- contains bands of known sizes
- used to compare DNA fragments against bands of known sizes to determine size
(since conditions affect separation of fragments, so each time process is done, separation will be slightly different)
factors that affect the rate at which DNA moves through a gel
- size of molecule (larger molecule moves slower)
- charge of molecule (increasing negative charge of DNA makes it move faster to positive electrode)
- voltage of power source (increase in electric current causes DNA to be more strongly attracted to positive electrode and move faster)
- concentration of agarose gel (denser- gel pores are smaller, move slower)
- temperature of environment (higher- more kinetic energy, move faster or increase in temp makes gel more fluid or could denature sample)
- pH of buffer (creates current, alters charge in DNA and changes attraction to electrode (check!))
- length of time (too long, DNA falls off)
- length of gel (bigger gel- can run it for longer)
What is DNA profiling?
- DNA fingerprinting
- individuals can be identified and compared via respective DNA profiles
- relies on hypervariable regions (regions of DNA that vary greatly between individuals) such as STRs
- short tandem repeats (non coding regions, 2-6 bases long)
- each individual has 2 copies of each str (one from each pair of homologous chromosomes)
advantages of using bacterial plasmids as vectors for gene cloning
- small size allows easy manipulation
- Carry a range of restriction enzyme sites
- Self-replicate
- Replicate at a faster rate than chromosomal DNA
- Have a variety of markers (e.g. antibiotic resistance or lacZ expression / fluorescence) allows for selection of transformed individuals
Name five types of enzymes required to create recombinant DNA. Explain the role of each enzyme in creating a recombinant plasmid containing the human insulin gene.
Endonuclease: isolate gene of interest by cleaving the phosphate sugar backbone by identifying specific recognition sites, creating sticky ends. same enzyme cleaves plasmid DNA, leaving same sticky end fragments.
DNA ligase: join the sugar-phosphate backbone between the fragments (catalysing formation of phosphodiester bonds)
Reverse transcriptase: catalyses single stranded RNA into single stranded complementary cDNA
DNA polymerase: uses single stranded cDNA as a template to build doube stranded cDNA
Taq polymerase: involved in creating large quantities of gene of interest to be inserted into plasmid by using PCR
Traditional PCR amplification of circular plasmids results in linear fragments. Suggest why this occurs. (2 marks)
- PCR involves specific primers binding (annealing) to specific DNA sequence.
- Taq polymerase extends a complementary strand with free nucleotides, forming a linear DNA fragment.
- start and end of the new fragment cannot ligate to form phosphodiester bonds at the sugar phosphate backbone. The new DNA fragment will remain linear.
By referring to the specific structure of the DNA double helix, outline why both a forward and reverse primer are required in the PCR process.
- two strands of DNA are antiparallel
- DNA is always synthesised 5’ to 3’
- primers must bind to 3’ end of template strand so Taq Polymerase can add complementary nucleotides to 3’ end of primer
Describe the specific property of the agarose gel that facilitates the movement of DNA fragments.
It is porous – small holes in the gel allow DNA fragments to move through.
What is the purpose of the running buffer that covers the gel?
It contains ions that carries an electric current through the gel, allowing the movement of DNA.
- maintains appropriate pH
What is a dye front?
- allows scientists to determine how far samples have moved through the gel
- dye will be the smallest sized component and therefore migrates faster than DNA
- all DNA fragments will be larger than the dye molecules so scientists will know they have not run off the gel.
also
- allows for bands to be seen under UV light
Why are regions of DNA that code for certain proteins and enzymes unsuitable for DNA profiling?
- code for products or functions that are the same for everyone
- In DNA profiline, scientists need unique patterns to identify an individual
What is the name for the non-coding regions of DNA that are studied in DNA profiling?
Short tandem repeats (2–6 base pairs long)
Variable number tandem repeats (20–60 base pairs long)
Why are endonucleases often referred to as restriction enzymes?
Endonucleases are enzymes that cut at specific recognition sites. These sites are often referred to as ‘restriction sites’. Therefore, restriction enzymes are enzymes that cut DNA at restriction sites to form fragments of smaller sizes.
List 3 applications of dna profiling
crime scene analysis
paternity testing
identification of bodies after disasters
What is the purpose of each of the following in gel electrophoresis?
i. An electric field
ii. agarose gel
iii. PCR
iv. endonucleases
i. allows the negatively charged DNA to move through the agarose gel towards the positively charged pole, allowing the fragments to move.
ii. allows different-sized fragments to be separated. As it is a porous substance, smaller fragments are able to more easily migrate through the gel
iii. allows the DNA collected to be amplified so there is sufficient DNA to see on gel and analyse
iv. Cut DNA into different sized fragments based on presence of recognition sites
is a DNA ladder always needed?
no
for example, when comparing samples to each other such as in paternity testing or crime scenes
Outline one ethical issue associated with the use of gene cloning in the manufacture of a pharmaceutical product.
- Who has legal ownership and how will the product be made available for all people in the world?
- Changing a species’ DNA may result in unforeseen consequences.
- Concern that the pharmaceutical product may contain bacteria that will cause disease.
- Not natural and therefore may be against religious/moral views.
explain why RNA must be converted into DNA in RT-PCR
DNA Polymerase, enzyme used in PCR, can only make copies of DNA.
PCR requires double stranded DNA template
What is the purpose of DNA amplification in PCR?
- PCR makes multiple identical copies
- large enough sample to be analysed
samples of DNA are originally extracted from yeast cells. Describe the processes DNA would be exposed to prior to its addition to the gel.
- DNA would need to be purified from cell
- DNA would need to be amplified to obtain sufficient amount of DNA to analyse
- DNA would need to be mixed with restriction enzymes to cut DNA into fragments
Outline 2 safety guidelines when doing gel electrophoresis:
- safety glasses should be worn to protect the eyes
- making sure electrophoresis equipment is turned off to avoid electrocution
- wearing gloves to avoid contamination from chemicals or DNA
- correct disposal of materials
What is a valid experiment?
those that test what they claim to test
Usual process before gel electrophoresis
- obtain DNA sample from cells
- target STR region and amplify if (PCR)
- cut DNA with restriction enzymes at either side of STR
- separate on gel
- Compare with other individuals
