Enzymes that manipulate DNA

Question 1

Define Endonuclease

Answer 1

An endonuclease is an enzyme that breaks the phosphodiester bond between two nucelotides in a polynucleotide chain.

Question 2

Define a recognition site

Answer 2

A recognition site is a specific target sequence of DNA upon which restriction endonucleases do their job.

Question 3

Define Restriction enzymes/endonucleases

Answer 3

Restriction endonucleases are any enzymes that acts like ‘molecular scissors’ to cut nucleic acid strands at specific recognition sites.

Question 4

Distinguish the difference between sticky and blunt end cuts of DNA.

Answer 4

Sticky end cuts refers to the result of a staggered cut through double-stranded DNA by an endonuclease resulting in overhanging nucleotides. Blunt end cuts refers to the result of a straight cut across the double-stranded DNA by the endonuclease resulting in no overhanging nucleotides.

Question 5

Define Ligases

Answer 5

Ligases refer to an enzyme that joins molecules, including DNA or RNA, together by catalysing the formation of phosphodiester bonds.

Question 6

Define a primer

Answer 6

A primer is a short, single strand of nucleic acids that acts as a starting point for polymerase enzymes to attach.

Question 7

Define a bateriophage

Answer 7

A bacteriophage is a virus that infects prokaryotic organisms.

Question 8

Explain what CRISPR-Cas9 is

Answer 8

CRISPR-Cas9 is a complex formed between gRNA and Cas9 which cuts a target sequence of DNA. BActeria use this for protection and used for editing genomes.

Question 9

Define spacers

Answer 9

Spacers are short sequences of DNA obtained from invading bacteriophages that are added into CRISPR sequence.

Question 10

Define protospacer adjacent motif (PAM)

Answer 10

PAM is a sequence of two-six nucleotides that is found immediately next to the DNA targeted by Cas9.

Question 11

Define guide RNA (gRNA)

Answer 11

Guide RNA is RNA which has a specific sequence determined by CRISPR to guide Cas9 to a specific location or site.

Question 12

Define single guide RNA (sgRNA)

Answer 12

Single guide RNA is guide RNA utilised by scientists to instruct Cas9 to cut a specific site when using CRISPR in gene editing.

Question 13

Identify the steps of using CRISPR-Cas9 in gene editing

Answer 13

1. sgRNA is created with a complimentary spacer to target DNA wanted to be cut. 2. Cas9 enzyme is obtained with the appropriate target PAM sequence. 3. Cas9 and sgRNA are added together and mixed to bind together and create CRISPR-Cas9 complex. 4. sgRNA-Cas9 mixture is then injected into a specific cell. 5. Cas9 finds target PAM sequence and checks to see whether the sgRNA aligns. 6. Cas9 cuts the selected sequence of DNA. 7. DNA has a blund end cut that cell will try to repair.

Question 14

Identify the applications of CRISP-Cas9 technology.

Answer 14

Some applications of this technology include: Research, Dealing with diseases and Agriculture.

Question 15

Define Differentiation

Answer 15

Differentiation is the process of which cells can develop specialised characteristics, typically transforming from one cell type to another specialised cell type.

Question 16

Define the Polymerase Chain Reaction (PCR)

Answer 16

The polymerase chain reaction is a technique used to produce many identical copies of DNA from initially a smaller sample.

Question 17

Define Taq Polymerase

Answer 17

taq polymerase is a heat-resistant DNA polymerase enzyme which amplifies a single-stranded DNA molecule by attaching complementary nucleotides.

Question 18

Identify the steps of Taq Polymerase

Answer 18

The steps of taq polymerase are Denaturation, Annealing and Elongation.

Question 19

Explain the process of Denaturation

Answer 19

DNA is heated up to approximately 90-95 degrees to break the hydrogen bonds between the bases and separate the strands, forming single-stranded DNA.

Question 20

Explain the process of Annealing

Answer 20

The single-stranded DNA is cooled to approximately 50-55 degrees to allow for primers to bind to complimentary sequences on single-stranded DNA.

Question 21

Explain the process of Elongation

Answer 21

The DNA is heated again to 72 degrees, which allows for Taq polymerase to work optimally. Taq polymerase binds to primer, acts as a starting point, and begins synthesising a new complimentary strand of DNA.

Question 22

Define Gel electrophoresis

Answer 22

Gel Electrophoresis is a technique that separates DNA fragments based on their size.

Question 23

Define a well

Answer 23

A well refers to an indent in the gel into which a DNA sample is loaded into.

Question 24

Define a standard ladder

Answer 24

A standard ladder refers to a mixture of DNA fragments of known length that are used to infer the size of the fragments to move through.

Question 25

Define agarose gel

Answer 25

Agarose gel refers to a sponge-like gel that contains pores for DNA fragments to move through.

Question 26

Define buffer

Answer 26

It refers to a solution that carries electrical currents through the agarose gel.

Question 27

Define a base pair (bp)

Answer 27

A base pair is a unit of measurement that corresponds to one nucleotide.

Question 28

Define DNA profiling

Answer 28

DNA profiling is the process of identification on the basis of an individuals genetic information.

Question 29

Define heterozygous

Answer 29

Heterozygous refers to having different alleles for the same gene on homologous chromosomes.

Question 30

Define homozygous

Answer 30

Homozygous refers to havign identical alleles for the same gene on homologous chromosomes.

Question 31

Define Short Tandem Repeats (STR)

Answer 31

short tandem repeats are short, repeated sequences of nucelotides found in the non-coding regions of nuclear DNA.

Question 32

Define a plasmid

Answer 32

A plasmid is a small, circular loop of DNA separate from the chromosome, found in bacteria.

Question 33

Define Recombinant plasmids

Answer 33

recombinant plasmids refer to a circular DNA vector that is ligated to incorporate a gene of interest

Question 34

Define the process of bacterial transformation

Answer 34

Bactral transformation is the process by which bacteria take up foreign DNA from their environment. It is used to introduce recombinant plasmids into bacteria.

Question 35

Define plasmid vector

Answer 35

Plasmid vectors refer to a piece of circular DNA that is modified to be an ideal vector for bactral transformation experiment.

Question 36

Define antibiotic resistance gene

Answer 36

The antibiotic resistance gene refers to the gene which has resistance to antibiotics.

Question 37

Define reporter gene

Answer 37

The reporter gene refers to the gene with ann easily identifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest.

Question 38

Define genetic engineering

Answer 38

Genetic engineering refers to the process of using biotechnology to alter the genome of an organism. It involves genes being silenced, inserted into a genome, removed from a genome or altered by replacing nucleotides.

Question 39

Define a GMO

Answer 39

A genetically modified organism refers to an organism with genetic material that has been altered using genetic engineering tech.

Question 40

Define cisgenic organisms

Answer 40

A cisgenic organism refers to a GMO thatc contains foreign genetic material from a sexually compatible donor organism, typically from same species.

Question 41

Define transgenic organisms

Answer 41

Transgenic organisms refer to a GMO that contains foreign genetic material from a separate species (or recombinant DNA from the same species that has been manipulated before introduction).

Question 42

Explain the use of GMO in agriculture

Answer 42

through the use of agriculture, the applications of GMO’s include: increased crop productivity and increased disease resistance of the crop.

Question 43

Define Transgene

Answer 43

Transgene refers to a gene that has been artificially introduced into the genome of a separate organism.

Question 44

Identify the issues surrounding the use of GMO’s

Answer 44

Some issues surrounding the use of GMO’s include: Biological implications - GM crops may lose effectiveness, widespread use of GM crops may result in loss of genetic diversity, Social implications - costly for farmers, strict packaging and marketing guidelines, Ethical implications - Some people consider GMO’s to be unnatural, people believe genetically modifying animals is inhumane and wrong.