Unit 4 SAC 3: Genetic Engineering

Question 1

What is a restriction enzyme?

Answer 1

Restriction enzymes are bacterial enzymes that recognise a short sequence of bases in a DNA molecule. Restriction enzymes cut at the specific recognition site with the result being the formation of a sticky end or blunt end.

Question 2

What is a recognition site?

Answer 2

A recognition site is a sequence of 3 to 6 nucleotides within a DNA molecule that forms the specific site for a restriction enzyme that can cut the DNA at that point.

Question 3

What is a sticky end?

Answer 3

Sticky ends are overhanging ends with exposed nucleotide bases at each. DNA cut in this way can be joined to other DNA with sticky ends (specific to their recognition sites).

Question 4

What is a blunt end?

Answer 4

Blunt ends are ends with no nucleotide bases exposed at each. DNA cut this way can be joined with other DNA with blunt ends, but as their are no recognise sites this is non specific.

Question 5

What is a polymerase chain reaction (PCR)?

Answer 5

PCR is a technique used to amplify a sequence of DNA in a test tube. PCR is a chain reaction of DNA replication events. At each cycle of synthesis, the number of copies doubles.

Question 6

What is dissociation?

Answer 6

Dissociation in a PCR, is when the double helix of DNA is separated into two single strands. This is done by by breaking the hydrogen bonds between strands by heating.

Question 7

What is hybridisation?

Answer 7

Hybridisation AKA annealing is the pairing between single-stranded complementary DNA segments.

Question 8

Steps in the PCR process? (PCR involves DNA replication)

Answer 8

Steps in the PCR process include:
1. Separate strands:
Separate the target DNA strands by heating to 98 for five minutes.
2. Add reaction mix:
Add primers (short RNA strands that provide a starting point for DNA replication) nucleotides and DNA polymerase enzyme.
3. Incubate:
Cool to 60 and incubate for few minutes. In this time primers are attaching to single stranded DNA allowing polymerase to synthesise complementary strands.

Question 9

Predict the number of fragments produced by a restriction enzyme.

Answer 9

• In linear DNA for example that of a human,
  x cuts = x fragments.
• In circular DNA for example that of a bacteria plasmid,
  x cuts = x+1 fragments

Question 10

What is gel electrophoresis?

Answer 10

Gel electrophoresis is a process which enables the separation of pieces of DNA using an electrical charge. The use of a standard or “ladder” enables the identification of different length DNA fragments.

Question 11

What are the steps in gel electrophoresis?

Answer 11

1. DNA is cut by a restriction enzyme.
2. DNA is loaded at the negative end of the gel slab along with molecular weight standards.
3. Slab is immersed into a buffer solution containing electrolytes.
4. Due to the negative nature of DNA, fragments are pulled towards the positive terminal at the opposite end of the wells.
5. The charge, along with the pores (holes) in the gel allow smaller molecules to move further in a set period of time.
6. DNA fragments observed with ultraviolet light.

Question 12

Explain how electrophoresis graphs/photos are interpreted.

Answer 12

Band patterns can be used to determine the DNA sequence and the protein that it codes for.

Question 13

What is a DNA probe and what it its purpose?

Answer 13

A DNA probe is a piece of single stranded DNA (or RNA) complementary in bases to its target DNA (the fragment of interest). The purpose of a DNA probe is to find a particular DNA fragment from amongst a large number of fragments. The process in which the probe is added simply involves the target DNA dissociating, the probe being added and the hybridisation occurring with the target.

Question 14

What is in situ hybridisation?

Answer 14

Hybridisation simply means the pairing between single-stranded complementary DNA segments. In Situ means in its place, therefore the DNA remains in the cell (in its place) while the probe is added.

Question 15

What is southern blot?

Answer 15

Southern blot involves using a probe to locate a DNA fragment in a gel after electrophoresis. Southern blot can only occur after a number of steps including:

1. The DNA being cut with restriction enzymes.
2. The fragments being separated after gel electrophoresis.
3. The DNA is transferred to Nitrocellulose filter paper.
4. Probes are added and hybridisation occurs with target DNA.
5. When viewed by auto radiograph, target DNA are located.

Question 16

What is reverse transcription and what is its purpose?

Answer 16

Reverse transcription is the process of making cDNA from an mRNA template. The purpose of reverse transcription is to create cDNA and use it to study the expression of genes in specific cells (only genes that are expressed transcribe into mRNA).

Question 17

What is cDNA?

Answer 17

cDNA is copy DNA that is synthesised from a mature mRNA template.

Question 18

What is reverse transcriptase?

Answer 18

Reverse transcriptase is the enzyme responsible for building cDNA along the mRNA template.

Question 19

What is a DNA sequence and what is the purpose of DNA sequencing?

Answer 19

A DNA sequence is an order of bases, the purpose of DNA sequencing is to determine the order of bases in a gene or segment of DNA.

Question 20

What are terminating nucleotides?

Answer 20

Terminating nucleotides are nucleotides which when added to a growing DNA chain, stop it.

Question 21

What is a tag in DNA sequencing?

Answer 21

A tag in DNA sequencing is a coloured fluorescent marker attached to a terminating nucleotide.

Question 22

What is DNA profiling and what is its purpose?

Answer 22

DNA profiling is a process that identifies DNA from different individuals by their short tandem repeats and micro satellites. The purpose of DNA profiling is to identify individual’s genetic differences for forensics and paternity.

Question 23

What are short tandem repeats.

Answer 23

Short tandem repeats are found in nuclear DNA and are unique to all individuals except twins. Short tandem repeats are regions of 2-5 base pairs long that are repeated at any locus on a chromosome.

Question 24

Explain the process of DNA profiling.

Answer 24

DNA profiling is used in fingerprinting and involves the following:

1. Restriction enzymes cut DNA into fragments.
2. Fragments amplified by PCR
3. Fragments put through gel electrophoresis and seen by hybridisation with probes
4. DNA bands are compared.

Question 25

What is a plasmid

Answer 25

A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell’s chromosomal DNA. The genes carried in plasmids often contain genetic advantages.

Question 26

What do plasmids do?

Answer 26

Plasmids are used in genetic engineering to reproduce recombinant genetic material. When a plasmid is inserted into a bacterium, the bacterium is encouraged to multiply, creating more copies of the recombinant DNA.

Question 27

Define Gene cloning.

Answer 27

Gene cloning is the production of large quantities of identical copies of a gene usually to use its product.

Question 28

Explain gene cloning.

Answer 28

The gene is inserted into a plasmid to form a recombinant plasmid. This is then added to a test tube along with bacterial cells that, after a shock treatment will take up some of the plasmids.

When the recombinant plasmid is made, a gene coding for antibiotic resistance is added so only bacteria that have taken up the plasmid are able to multiply on an agar plate that contains antibiotic.

Question 29

What is gene delivery?

Answer 29

Gene delivery is the process of inserting foreign DNA into host cells.

Question 30

Gene transformation.

Answer 30

Is the process of inserting foreign DNA into prokaryotic cells using plasmids.

Question 31

Gene transfection

Answer 31

is the process of introducing foreign DNA into eukaryotic cells.

Question 32

Vector

Answer 32

Agents such as plasmids or viruses, which carry DNA into a cell.

Question 33

Plasmid vector

Answer 33

Inserting a new piece of DNA into the circular plasmid.

Question 34

Bacteriophages as vectors

Answer 34

Viruses which infect bacteria, used to carry DNA into bacterial cells.

Question 35

Transgenic organism.

Answer 35

An organism carrying in its genome, one or more genes artificially introduced from another species.

Question 36

Explaining In Vivo Gene Cloning.

Answer 36

In Vivo gene cloning is the insertion of a gene into an organism and using the replication machinery of that organism to multiply the gene or produce its protein product.

Question 37

Name the steps in in Vivo gene cloning

Answer 37

1. Gene is isolated and introns removed + plasmid with specific recognition sites is used.
2. The human DNA and plasmid are treated with the same restriction enzyme for identical sticky ends and the DNA is the inserted into to the plasmid.
3. The DNA is added to a test tube along with bacteria that, after a shock treatment will take up some plasmids.
4. The bacteria are added to an agar plate with nutrients and antibiotic that allow only the bacteria that have taken up the plasmids to grow. This is due to a gene coding for antibiotic resistance being added earlier to our recombinant DNA.
5. The DNA can then be taken from the plasmid and amplified through PCR and separated through gel electrophoresis.