Genetic Engineeing

Question 1

Process of a gene being altered

Answer 1

1. isolation of the gene interest
2. isolation of genes into a carrier
3. transfer the vector to the organism that need to modified
4. Transformation of cells in the organism

Question 2

How can you obtain a cells DNA

Answer 2

The cells have to be lysed with chemicals, then the nucleus will be lysed and the DNA will be released

Question 3

Where are restriction enzymes naturally found

Answer 3

In bacterial cells

Question 4

Define Restriction enzyme

Answer 4

Found naturally in bacteria, they are used to cut DNA at a specific known recognition site.

Question 5

What types of ends can restriciton enzymes produce?

Answer 5

Sticky

Blunt

Question 6

Define Recognition Site:

Answer 6

A sequence of nucleotides that form a specific site for a restriction enzyme to cut DNA.

Question 7

Define Sticky Ends

Answer 7

A piece of DNA with that has been cut by restriction enzymes. The fragment will consist of a section of single stranded nucleotides, these fragments can form hydrogen bonds with a complementary strand.

Question 8

Define Blunt Ends

Answer 8

A double stranded piece of DNA that has been cut by a restriction enzyme.

Question 9

What is the calculation for linear DNA?

Answer 9

n cuts = n+1

Question 10

What is the calculation for circular DNA?

Answer 10

n cuts

Question 11

What is the role of DNA ligase

Answer 11

Catlayses the formation of covalent bonfd between sugar phosphate backbones of DNA fragments.

Question 12

Define Recombinant DNA

Answer 12

A DNA molecule made outside of the cell with segments from different organisms

Question 13

What is the key to recombinant DNA

Answer 13

The same restriction enzymes are used to cut open plasmids as to cut the desired DNA fragment this ensures there are complementary sticky ends.

Question 14

Explain the process of making plasmid recmobinant

Answer 14

1. Isolate the gene and cut with a restriction enzyme
2. Then cut the plasmid with the same restriction enzyme as the gene was cut, to ensure they are complementary
3. Mix together with ligase

Question 15

What do bactiera need to be in order to take up the plasmid?

Answer 15

Competent

Question 16

Explain why plasmids cant just go straight through the cell wall?

Answer 16

Plasmids have a neg charge
Cell wall/membrane has a negative charge
Therefore they repel eachother

Question 17

Why is CaCl2 added?

Answer 17

To make the cell membrane soften in order for holes to easily be formed in the heat shock process

Question 18

Why is there heat added in the process of bacterial transformation?

Answer 18

Heat shock makes holes

Question 19

Define the process of gel electrophroesis

Answer 19

The process of seperating DNA fragments accordance to their size.

Question 20

Which end of the agarose gel is the DNA attracted to?

Answer 20

Positive

Question 21

Explain how the agarous gel works

Answer 21

It is highly pourous and acts like a sieve where the shortest DNA fragments are able to move faster through the pores, therefore it is harder for the larger fragments to fit through the pores and they lag behind.

Question 22

What is the purpose of the buffer

Answer 22

• Allows for a stable environment including pH
• Allows for a temperature in that the agarose will not melt
• Contains ions which all for the electrical current to occur

Question 23

Define Plasmids

Answer 23

A vector that consists of a circular loop of DNA. A plasmid will carry DNA from one organism to another/

Question 24

Why are plasmids so useful

Answer 24

• Due to their circular shape and being small in size this causes plasmids to generally be harder to break
• Easy to manipulate
• Used for cloning they are replicated and divide when a bacteria does also

Question 25

What is the purpose of the marker?

Answer 25

Acts as a standard to indicate how far the DNA have travelled

Question 26

What must DNA be prepped with before undertaking electrophoresis

Answer 26

Glycerol must be added to ensure the DNA sinks into the well

A methlyline blue stain is added to track the overall migration of the DNA

Question 27

What is the role of the arabanose sugar?

Answer 27

Required for flurosence to occur

Question 28

Define competent

Answer 28

Treated in such a way that they are able to accept a plasmid vector

Question 29

What is reverse transcriptase used for?

Answer 29

Synthesises Dna from an RNA template

-Performs the reverse of transcription

Question 30

What is the process of microinjections

Answer 30

Involving the use of a microneedle to insert DNA into a living cell
Penetreates cell membrane and sometimes even nuclear membrane
used in cloning

Question 31

What is a probe

Answer 31

Short single stranded piece of DNA

makes DNA visible

Question 32

Explain briefly the process of southern blotting

Answer 32

1. DNA placed in seperate wells and a restriction enzyme was added to each to produce DNA fragments
2. Then underwent electrophoresis
3. DNA is transferred to paper and it is denatured in the process (single strands)
4. Placed then in a solution of radioactive probes

Question 33

What is PCr

Answer 33

technique used to amplify particular DNA fragments

Question 34

Why is PCR needed?

Answer 34

Many engineering genetic process require large amount of DNA

Question 35

Explain the 4 steps in PCR

Answer 35

1. Heat DNA to 95 degrees, DNA will denature and will form two
   strands
2. DNA is then made the temperature of 55 degrees and primers will joins to the two strands via comp base pairing
3. DNA heated to 72 degrrees and DNA polymerase intiates DNA synthesis
4. Process is repeated.

Question 36

What is DNA profiling

Answer 36

Using DNA to identify an individual

Question 37

What are some benefits of DNA profiiling

Answer 37

• Sensitive
• Requires small qunaities of DNA
• Fragments difffers in size
• Hours to perform

Question 38

What is DNA sequencing

Answer 38

Determining the sequence of nucleotides making up the lenght of DNA

• Restriction enzymes digest the strand
• Fragments isolated via gel electrophoresis
• sSequnece is determines by PCR or chemical analysis