DNA-centered applications

Question 1

Do all life forms use the same genetic code

Answer 1

Yes

Question 2

Explain how this made genetic engineering possible.

Answer 2

Because all organisms use the same DNA code, genes can function across different species. The discovery of restriction enzymes and ligases allowed scientists to cut and join DNA at specific sites, creating recombinant DNA. This made it possible to combine genes in new ways, enabling the design of new gene regulation and protein sequences.

Question 3

Describe the action of restriction enzymes.

Answer 3

Restriction endonucleases cleave double-stranded DNA in a sequence-specific manner at cut sites that have specific sequences that are normally palindromic.

Question 4

Describe the action of DNA ligases.

Answer 4

1. T4 ligase4 attaches cut DNA together.
2. The new covalent bonds formed by the action of bacteriophage T4 ligase allow two unrelated DNA duplexes to be combined to form a single continuous duplex.

Question 5

Explain how this allow DNA from different sources linked together.

Answer 5

Two pieces of DNA cut with SfuI have matching
sticky ends that can base-pair (form hydrogen bonds ) to each other =
Annealing the ligation

Question 6

Define a bacterial plasmid.

Answer 6

A circular double-stranded DNA that is separate from the genomic DNA of a cell.

Question 7

Describe how plasmids can be used to carry non-bacterial DNA (such as ours) in bacteria.

Answer 7

A DNA sequence of interest is cut using restriction enzymes and inserted into a plasmid with matching ends using ligase. The recombinant plasmid is then introduced into bacteria, where it replicates, allowing many copies of the non-bacterial DNA to be produced and stored.

Question 8

Explain in very general terms how this allows us to obtain many copies of a particular sequence of DNA by “molecular cloning”.

Answer 8

1. DNA cloning can be undertaken in E.coli.
2. Protein-coding DNA sequences are cut
   and ligated with plasmid DNA so that
   they can be incorporated into bacteria.
3. With the right signals, the bacteria can
   make the human protein in the same
   way as it makes its own proteins = producing human proteins

Question 9

Describe the kind of enzyme that is needed for PCR.

Answer 9

Taq polymerase = Heat-stable DNA polymerase

Question 10

Explain the role of primers in PCR.

Answer 10

Specific segments of duplex (double-stranded) DNA are copied to make two new duplexes exactly like the one started with = polymerization (DNA synthesis)

Question 11

Describe the process of PCR and how a large amount of a single sequence can be obtained.

Answer 11

1. Duplex is heated to separate the two DNA strands.
2. Two primers are allowed to base pair
   with the strands.
3. Taq polymerase synthesizes DNA starting from each primer.
4. Two copies of the specific DNA
   segment are produced.
5. DNA is heated again to separate the
   strands.
6. This process repeats again and can continue up to 25 or more times through heating and cooling, resulting in large amounts of DNA

Question 12

Name one important medical test that is done using PCR, and how does it work?

Answer 12

PCR is used for testing SARS-CoV-2 viral RNA
1. Viral RNA is collected by nasal swabs
2. Reverse transcribes make DNA from RNA = cDNA
3. primers base pair with DNA at the edges of a segment of the viral sequence
4. A fluorescent probe is used to measure the amplified DNA product
5. (If patient is negative = no amplification)

Question 13

Indicate how the amplified DNA is detected in the medical test.

Answer 13

The abundant ”amplified” DN product can be detected by agarose electrophoresis followed by staining

Question 14

Define precision medicine

Answer 14

Medical care that is targeted to an individual and that is based on their specific genetic
characteristics

Question 15

Give an example of precision medicine dependent upon genetic testing in patient care.

Answer 15

Children with cardiomyopathy may have genetic mutations (DNA base changes) that can
be identified through genetic testing.

Question 16

Describe the difference between sticky and blunt ends

Answer 16

Sticky (overhanging) ends form when one DNA strand extends beyond the other. These ends only base-pair with DNA having the same matching end. Whereas Blunt ends have no overhang and match all other blunt ends = straight line

Question 17

What enzymes leave sticky and blunt ends

Answer 17

1. EcoR1 leave sticky = E.coli
2. Alu leave blunt ends = Arthrobacter luteus

Question 18

Define Genome

Answer 18

All the DNA in an individual (or in a particular
tissue or cell)

Question 19

Define Exome

Answer 19

All the expressed genes (copied from mRNA) in an individual (or in a particular tissue or cell)