DNA manipulation techniques and applications

Question 1

Ligase

Answer 1

An enzyme that catalyses the joining of double stranded DNA fragments at their sugar phosphate backbones → forms strong covalent (phosphodiester) bonds

Question 2

Endonuclease

Answer 2

Act like molecular scissors to cut or cleave DNA at specific recognition sequences (restriction sites)
Can produce sticky or blunt ends
Sticky ends results in a jagged cut
Blunt end results in a straight cut

Question 3

Polymerase

Answer 3

Enzyme involved in synthesising nucleic acids
Assembles DNA in a 5’ to 3’ direction once double helix is unzipped

Question 4

Reverse transcription

Answer 4

Objective - make photocopy of genetic material with exons only (much shorter) Can be used in DNA technology - can put in small vector (viral, fungi, yeast)
Isolate mRNA strand and work backwards to create DNA molecule with reverse transcriptase
Use DNA polymerase help facilitate free floating nucleotides
New DNA molecule would only have exon sections

Question 5

Guide RNA

Answer 5

RNA sequence complementary to gene of interest that provides the platform for the Cas9 protein to bind to synthesis DNA

Question 6

Cas9

Answer 6

cleaves DNA at specific base sequences.

Question 7

PAM

Answer 7

Protospacer adjacent motif
A specific sequences of nucleotides that binds to the gene of interest and acts as a recognition molecular for the Cas9 to identify the gene location

Question 8

CRISPR-CAS9 natures way (bacteriophages)

Answer 8

1. Bacteriophage virus lands on a cell and injects its DNA
2. Viral genome gets incorporated into CRISPR region
3. When viral DNA is transcribed to mRNA, it becomes guide RNA and forms a complex with Cas 9 enzyme which float around in the cell with the RNA
4. When the same virus reinjects its DNA into the cell, Cas 9 have complementary RNA and will cut up viral DNA

Question 9

CRISPR-Cas9 application

Answer 9

1. Scientists manufacture cas9 protein, guide RNA and PAM- specific to the target DNA
2. The cas9 will identify the PAM sequence and guide RNA directs it to cut both strands of DNA
3. Scientists can insert a new piece of DNA (donor DNA) at the site of the cut. They may also remove or replace sections of DNA or add and delete nucleotides
4. The cell detects and repairs the broken strand of DNA. When the DNA is repaired any changes made are integrated into the genomic DNA.
   → knock out, introduce, edit, activate/repress genes

Question 10

PCR

Answer 10

1. Denaturing - double stranded piece of DNA is heated to approx 94°C, causing the hydrogen bonds between the complementary strands to break.
2. Annealing- temp is lowered to approximately 55 °C for two minutes. Primers are added and anneal to the complementary regions of the DNA strand. Primers bind to the target DNA sequences at 3′ ends and initiate DNA synthesis (polymerisation).
3. Extension - temp is raised to approx 72°C and taq polymerase synthesises a new strand of DNA (using the primers as a starting point) by adding free nucleotides.
   This process is repeated, doubling the DNA each time.

Question 11

Gel electrophoresis

Answer 11

1. Restriction enzymes are used to cut DNA strands into fragments combined with DNA loading dye (that makes the DNA glow under UV)
2. DNA is placed into a well at one end of a piece of agarose gel (that is immersed in a buffer solution - to maintain pH)
3. Electrodes are attached to each end of the gel (positive at the far end and negative at starting origin) and an electric current passes through gel
4. As DNA molecules are negatively charged DNA pieces travel from the negative to positive end of the gel.
   –> smaller ones travel further

Question 12

Bacterial plasmid for insulin

Answer 12

1. The selection marker gene from the plasmid is cut using a specific endonuclease in order to create sticky ends. PCR is carried out to amplify the DNA
2. The insulin gene is prepared using the same endonuclease so that the desired gene has sticky ends complementary to the cut plasmid. (this is done with 2 separate insulin genes for chain A and B)
3. The insulin genes and the plasmid vector are mixed with DNA ligase, and, in some cases, their sticky ends may anneal and form a covalent bond at the sugar phosphate backbone
4. The culture is incubated, replicating every 20 mins
5. The two chains are then mixed together and re-join by forming disulfide bonds

Question 13

Bacterial pasmid

Answer 13

1. The selection marker/reporter gene from the plasmid is cut using a specific cutting enzyme (endonuclease) in order to create sticky ends. PCR is carried out to amplify the DNA
2. The desired gene is prepared using the same endonuclease so that the desired gene has sticky ends complementary to the cut plasmid.
3. The desired fragments and the plasmid vector are mixed, and, in some cases, their sticky ends may anneal
4. The joining enzyme, DNA ligase, is added and this makes the joins permanent through covalent bonding at the sugar phosphate backbone

Question 14

Heat shock technique

Answer 14

Rapidly increasing the temperature to 42 degrees and decreasing the temperature to 0 degrees to increase the membrane permeability in order to enhance the likelihood of bacterial transformation

Question 15

Antibiotic selection

Answer 15

Used to check if the vector cell took up the recombinant plasmid. Bacteria species use a plasmid that has the antibiotic resistant gene and this will confirm if the vector cell has the recombinant plasmid.

Question 16

GMO

Answer 16

Organisms whose genomes have been altered using genetic engineering technology in which the change is heritable (add or silence a gene)

Question 17

Transgenic organisms

Answer 17

Subgroup of GMOs in which the alteration to the genome involves the genetic material from a different species

Question 18

Applications

Answer 18

Increase crop productivity, herbicide resistant crops, natural pesticide and insecticides