demo 2 Flashcards
(12 cards)
What are the roles of polymerases, ligases, and endonucleases in DNA manipulation?
Polymerase: Synthesizes DNA strands by adding nucleotides in a 5’ to 3’ direction; used in PCR to amplify DNA.
Ligase: Joins fragments of DNA by forming phosphodiester bonds; essential in DNA recombination.
Endonucleases (Restriction enzymes): Cut DNA at specific recognition sites, producing blunt or sticky ends; used in gene cloning and genetic engineering.
If the restriction site is cut in the centre, the restriction enzyme leaves ‘blunt ends’. If it is cut in
any other location, the resulting ends are ‘sticky ends’
CRISPR
CRISPR arrays contain (partially) palindromic sequences,
punctuated by spacers, which are DNA captured from bacteriophage
viruses that have previously infected the cell.
To make a spacer, enzymes
(Cas1 and Cas2) cut out a ‘protospacer’ upstream of a PAM sequence in the viral DNA.
The CRISPR array is transcribed into a single pre-crRNA molecule. TracrRNA anneals to the
repeats in the CRISPR array. RNAse enzymes cut the pre-CRISPR to make cr:tracrRNA, which
becomes a gRNA when it is picked up by a Cas9 enzyme.
BACTERIAL DEFENCE AGAIN VIRAL INVASION
1.virus invades bacterial cell
2. new spacer is deprived from virus and integrated in CRISPR sequence
3. CRISPR RNA is formed
4. CRISPR RNA guides molecular machinery to target and destroy viral genome.
Cas9
Cas9 searches DNA for a PAM, and upon finding it, compares the sequence of bases in the
AMPLIFYING DNA fragments –
Polymerase Chain Reaction (PCR) steps
STEP 1: Denaturation
Double-stranded DNA sample is DENATURED –
separated/dissociates into single (complementary) strands
The weak hydrogen bonds that usually hold the two
complementary strands together at normal temperatures are
disrupted.
Results in two single stranded DNA strands.
2min at 94-95 °C
STEP 2: Annealing (bind
primers)
The mixture is cooled to 55 °C (50-600C) for 2min
Artificially synthesised short segments of single-stranded DNA (primers), are
added; these primers pair (anneal) with regions at either end of the DNA
region of interest (hydrogen bonds form).
Synthesis will be in the 3’ to 5’ direction(new strand). NB each strand is anti
parallel and the 3’ end is where the primer with BIND (see next slide)
primers will be in forward and reverse sequence
STEP 3: Extension
DNA polymerase uses primers as a starting point
and extends them so that TWO complete double
strands are formed.
The most common enzyme for this step
is Taq polymerase,
standard ladder
A standard ladder of DNA fragments of known sizes is usually
run through the gel at the same time as the unknown DNA
samples. In some cases, this is referred to as an allele ladder.
The size of unknown DNA fragments can be approximated by
comparing the positions of their bands with those of the known
standards.
NB: ALWAYS check for this standard when analysing a gel.
How does gel electrophoresis separate DNA fragments?
DNA is placed in wells of an agarose gel and subjected to an electric field.
DNA is negatively charged and moves towards the positive electrode.
Smaller fragments travel further, while larger fragments move slower.
Uses: DNA profiling, crime scene analysis, paternity testing
Plasmids: VECTORS of DNA
A VECTOR is a CARRIER
If a plasmid has been engineered to contain a specific piece of DNA
it becomes a ‘vector’
A SELECTABLE marker gene: Used to screen for transformed
bacteria which have taken up the plasmid; codes for an easily
identifiable trait.
Eg: Antibiotic resistance: Bacteria containing the plasmid with
the marker can be selected from among other bacteria by
exposure to the antibiotic.)
A SCREENING/REPORTER marker gene: Used to screen for
transformed bacteria which have taken up the gene of
interest; codes for an easily identifiable trait.
creating recombinant plasmids steps involved
Step 1: Choosing a restriction enzyme
An endonuclease is chosen to cut upstream and downstream of the gene, leaving sticky ends.
Step 2: Choosing a plasmid
A plasmid is chosen that has two genes that each encode observable traits. Of these genes,
one must contain the restriction site of the restriction endonuclease being used.
Step 3: Using the restriction endonuclease
The same restriction enzyme is used to cut both the source gene, and the plasmid. As a
result, the source gene and the plasmid have complementary sticky ends.
Step 4: Making a recombinant plasmid
When the source gene and the plasmids are mixed, sometimes the source gene will incorporate
into the plasmid, creating a ‘recombinant’. The bond is completed by adding ligase
When the source gene and the plasmids are mixed, sometimes the source gene will incorporate
into the plasmid. A plasmid containing foreign ’passenger’ DNA is called ‘recombinant’.
Step 5: Transformation
Bacteria (such as E. coli) are made competent to take up a plasmid. Only some bacteria will
take up a plasmid. Only some of those will take up the recombinant plasmid.
What is the difference between genetically modified organisms (GMOs) and transgenic organisms?
Genetically Modified Organisms (GMOs): Organisms whose DNA has been altered using genetic engineering.
Transgenic Organisms (TGOs): A subtype of GMOs that contain genes from a different species.
All TGOs are GMOs, but not all GMOs are transgenic.
How are GMOs used in agriculture?
Herbicide-resistant crops (e.g., Roundup Ready soybeans)
Contain a gene that makes them resistant to glyphosate, allowing farmers to kill weeds without harming crops.
Pest-resistant crops (e.g., Bt cotton & Bt corn)
Produce a natural bacterial toxin (Bt toxin) that kills insect pests.
Reduces the need for chemical insecticides.
Disease-resistant crops
Some GM bananas are resistant to Panama disease, a fungal infection that threatens global banana production.
