Manipulating DNA - Unit 3 AOS 1

Question 1

endonucleases (restriction enzymes)

Answer 1

• an enzyme that breaks phosphodiester bonds between two nucleotides in a polypeptide chain
• they cut the sugar-phosphate backbone at specific recognition sites
• they naturally occur in bacteria and can act as a defence mechanism against invading bacteria

Question 2

sticky end

Answer 2

• the result of a staggered cut through DNA
• overhanging nucleotides
• they are sticky because they will be attracted to unpaired complementary nucleotides
• advantageous becuse DNA will be oriented the righ way

Question 3

blunt end

Answer 3

• the result of a straight cut across DNA, resulting in no overhanging nucleotides

Question 4

Ligases

Answer 4

• enzymes that join two pieces of DNA at their sugar-phosphate backbone, catalysing the formation of a phosphodiester bond

Question 5

polymerases

Answer 5

• These enzymes synthesize new strands of nucleic acids (either DNA or RNA) by adding nucleotides to a pre-existing chain, using a template strand to guide the sequence.
• DNA polymerase synthesis DNA molecules from nucleotides
• RNA polymerase synthesises RNA molecules from a strand of DNA
• they require a primer

Question 6

Primer

Answer 6

• a short stand of nucleic acids which acts as a starting point for polymerase enzymes to attach to.
• they are complementary to the template strand

Question 7

CRISPR Cas9

Answer 7

• a complex formed between Cas 9 and gRNA which is found in bacteria

Question 8

Cas 9

Answer 8

• an endonuclease that cuts DNAat a point determined by gRNA

Question 9

gRNA

Answer 9

• a specific sequence of RNA which recognises the desired DNA and directs the Cas 9 to cut the DNA

Question 10

protospacer

Answer 10

• a short sequence
  of DNA extracted from a
  bacteriophage by Cas1 and Cas2,
  which has yet to be incorporated
  into the CRISPR gene

Question 11

Spacer

Answer 11

• short sequences of
  DNA obtained from invading
  bacteriophages that are added into
  the CRISPR sequence

Question 12

protospacer adjacent motif (PAM)

Answer 12

• a sequence of two-six nucleotides
  that is found immediately next to
  the DNA targeted by Cas9
• cas 1 and cas 2 search for a PAM sequence and cut out a photospacer just before it so the PAM sequence is not included in the photospacer.
• cas 9 also searches for a pam sequence next to the targen DNA so that is doesn’t need to search through every sequence of DNA
• PAM sequences are never found in bacterias own CRISPR repeats to that Ca9 9 verver cuts its own DNA

Question 13

CRISPR Cas 9 defence system

Answer 13

• exposure - the bateriophage injects some of its DNA into the bacteria. CAS 1 and CAS 2 cut out a protospacer which is introduced to the genome to become a spacer
• expression - the CRISPR spacers are transcribed and converted into gRNA. gRNA binds directly to Cas 9 creating a CRISPR cas9 complex. This is directed to any viral DNA that is complementary to the gRNA
• extermination - Cas 9 cuts the sugar phosphate backbone inactivating the virus

Question 14

repair mechanisms

Answer 14

• when the viral DNA is cut, the bacterium will want to repair it
• mutations in the repair mechanisms will inactivate the virus
• the whole process will continue until a mutation occurs to inactivate the virus.

Question 15

genetic modifications

Answer 15

• the manipulation of an organism’s genetic material using biotechnology
• removing or replacing DNA within a genome
• it can be used to fix deleterious mutations which negatively affect the organism

Question 16

gene therapy

Answer 16

• repairing genetic mutations by replacing a defective gene with a healthy one

Question 17

sgRNA

Answer 17

• guide RNA utilised by scientists to instruct Cas9 to cut a specific site
  when using CRISPR-Cas9 in gene editing

Question 18

using CRISPR cas 9 in gene editing

Answer 18

• Synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA that scientists wish to cut.
• A Cas9 enzyme is obtained with an appropriate target PAM sequence
• Cas9 and sgRNA are added together in a mixture and bind together to create the CRISPR Cas 9 complex
• The sgRNA-Cas9 mixture is then injected into a specific cell, such as a zygote.
• The Cas 9 finds the target PAM sequence and checks whether the sgRNA aligns with
  the DNA.
• Cas9 cuts the selected sequence of DNA
• The DNA has a blunt end cut that the cell will attempt to repair.
  8 When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site.
• Scientists may inject particular nucleotide sequences into the cell with the hope that it will ligate into the gap.

Question 19

gene knockout

Answer 19

•  a technique in gene editing where scientists prevent the expression of a target gene to understand its function in an organism

Question 20

gene knockin

Answer 20

• a technique in gene editing where scientists substitute or add nucleotides in a gene

Question 21

ethical implications to CRISPR Cas 9

Answer 21

• scientists must treat an embryo prior to it differentiating
• scientists cannot get consent from embryos to alter their genes
• ## equity of access

Question 22

polymerase chain reaction

Answer 22

• an artificial method of amplifying DNA in lab conditions
• it replicated DNA by making multiple identical copies.
• it is used by scientists when there is an insufficient amount of DNA

Question 23

steps of the polymerase chain reaction

Answer 23

• denaturing - the DNA is heated to approximately 95 degrees so that the hydrogen bonds between base pairs will break, separating the two stands
• annealing - the DNA is heated to approximately 55 degrees allowing primers to bind to complementary sections of the single stands of DNA.
• elongation - the DNA is heated back up to 72 degrees which is the optimal temperature of taq polymerase, allowing it to bind to the primers and begin synthesising two new stands of DNA
  -the process is then repeated until enough DNA has been amplified.

Question 24

taq polymerase

Answer 24

• a heat resistant DNA polymerase isolated from the bacteria thermus aquaticus
• it binds to primers during the annealing stage and begins synthesising a new complementary stand of DNA
• it is used instead of human DNA polymerase because it can withstand high heat without denaturing

Question 25

primers

Answer 25

• primers are used to bind to the stands of DNA allowing taq polymerase to function
• a forward primer binds to the 3 prime end of the template stand and synthesises a new stand in the same direction as RNA polymerase would function
• a reverse primer binds to the 3 prime end of the coding stand, synthesising a new stand of DNA in reverse.

Question 26

gel electrophoresis

Answer 26

Preparing the Gel: A gel, typically made of agarose, is placed in a gel box filled with a buffer solution that helps conduct electricity.

Loading the Samples: DNA (or RNA/proteins) samples are mixed with a dye and loaded into wells at one end of the gel.

Electric Current: An electric current is applied across the gel, with a positive charge at one end and a negative charge at the other. Since DNA is negatively charged (due to its phosphate backbone), it moves towards the positive end.

Separation by Size: Smaller DNA fragments move faster and further through the gel than larger ones, which results in bands forming at different distances from the wells.

Visualizing the Results: Once the run is complete, the gel is stained with a dye like ethidium bromide (which binds to DNA) and viewed under UV light to see the bands, representing different fragment sizes.

Question 27

agarose gel

Answer 27

• a gel that contains pores for the DNA to move through

Question 28

buffer

Answer 28

• a solution containing ions which carries an electrical current through the gel

Question 29

standard ladder

Answer 29

• a mixture of DNA fragments of known length that are used as a comparison

Question 30

controlled variables in gel electrophoresis

Answer 30

• voltage of the power source
• percentage of agarose in the gel (agarose is a polysaccharide added to the gel to control
  the rate at which fragments move through the gel)
• concentration of the solution (a ‘buffer’) covering the agarose gel
• temperature of the environment
• pH of the buffer solution
• length of time the sample of DNA is allowed to run for in the gel
• length of the gel (distance between the negative and positive electrodes).

Question 31

factors impacting the movement of DNA

Answer 31

• voltage - the higher the voltage, the faster the DNA will move
• gel composition - gels with greater density and agarose concentration will be harder for DNA to move through
• buffer concentration - the greater the concentration of ions in the buffer, the greater the electrical current
• time - the longer the DNA is left, the more it will travel

Question 32

DNA profiling

Answer 32

• a method of DNA analysis where regions of DNA from different individuals are analysed and compared
• coding regions of DNA are not useful because they code for gene products that everybody has
• STRs are small sections of repeated
  nucleotides that vary in length between people and are found in the non-coding areas
  of autosomal chromosomes
• STRs can vary significantly between individuals
• several variable number tandem repeats are studies so that there is a very low chance of two individuals having the exact same number of nucleotide repeats.

Question 33

heterozygous and homozygous in DNA profiling

Answer 33

• if an individual is heterozygous for an STR, they will have two bands
• if an individual is homozygous for an STR, they will have one thicker band

Question 34

making a recombinant plasmid

Answer 34

• the gene that scientists want to be expressed in recombinant plasmids (gene of interest) will be inserted into a vector
• it does not have introns

Question 35

plasmid vector

Answer 35

• a piece of circular DNA that is modified to be an idea vector for bacteria transformation
• i site on the plasmid can be recognised and cut by a restriction endonuclease
• DNA ligase is then needed to join the sugar phosphate backbone

Question 36

transforming bacteria

Answer 36

• recombinant plasmids are inserted into the cytoplasm of bacteria (bacterial transformations
• heat shock involves rapidly increasing and decreasing the temperature to increase membrane permeability
• electroporation involves delivering electric shocks to bacteria membranes to increase its permeability

Question 37

antibiotic selection

Answer 37

• to determine between transformed and untransformed bacteria, the mixture is cultured on an agar plate
• the untransformed bacteria will be killed off because it doesn’t contain the gene necessary for antibiotic resistance.

Question 38

making insulin - making the recombinant plasmids

Answer 38

• plasmid vectors prepared which contain the amp gene and tet gene to code for antibiotic resistance. tet gene has a recognition site to one of the restriction endonucleases
• two plasmids are used (A and B). EcorI and BamHI are used to cut the insulin genes and the plasmids, creating sticky ends. This means that the insulin should be complementary to the cut space in the vector and the two restriction enzymes means the insulin will co in the correct orientation
• DNA ligase completes the sugar phosphate backbone, creating recombinant plasmids.

Question 39

making insulin - bacterial transformation

Answer 39

• the plasmids are mixed with E.coli
• to determine which bacteria has taken up plasmids, they are grown on a plate containing ampicillin. the bacteria that grows, has a plasmid
• to determine which ones are recombinant, they are grown on a plate containing tetracycline. the ones that don’t grow, contain recombinant plasmids.

Question 40

genetic engineering

Answer 40

• the process of using biotechnology to alter the genome of an organism

Question 41

genetically modified organism

Answer 41

• an organism that has had its genome altered through genetic engineering technology

Question 42

cisgenic organism

Answer 42

• a genetically modified organism that has DNA from the same species inserted into it

Question 43

transgenic organism

Answer 43

• a genetically modified organism that has material from a different species inserted into its genome (recombination)

Question 44

GMOs in agriculture

Answer 44

1. a gene of interest with desirable characteristics is isolated - either synthetically or it is taken from an organism using restriction endonucleases
2. the gene is amplifies using PCR and inserted into a plasmid vector and then inserted into e recipient organism
3. the transformed and recombinant plasmids are identified using antibiotic resistant genes or fluorescent genes.
4. the transformed cells are then grown into GMO organisms

Question 45

benefits of GMO in agriculture

Answer 45

• increased crop productivity: improving the crop yield and the quality of crops (nutritional value and ability to grow in certain conditions
• increased disease resistant - genes can be inserted into crops that make them less impacted by harmful pathogens

Question 46

Gel electrophoresis steps

Answer 46

• samples containing DNA fragments and a die are loaded into wells at the negative end to the gel.
• the gel is immersed into a buffer solution containing ions so an electric charge can pass through
• a current passes through the gel using two electrodes. The DNA moves from the positive end to the negative end.
• smaller fragments move further
• at the end, a different dye that attaches to DNA is used so that the bands can become visible.