Recombinant DNA technology

Question 1

Why is mRNA often easier to obtain?

Answer 1

Most cells only contain two copies of each gene but many copies of the complementary mRNA.

Question 2

What does reverse transcriptase do?

Answer 2

makes DNA from an RNA template.

Question 3

What is the DNA produced from reverse transcriptase called?

Answer 3

cDNA (complementary DNA)

Question 4

What is a palindromic sequence?

Answer 4

One that reads the same in opposite directions.

Question 5

What can restriction endonucleases do?

Answer 5

Cut DNA at specific complementary palindromic sequences.

Question 6

What types of cut do restriction endonucleases make?

Answer 6

Sticky ends or straight.

Question 7

How does a gene machine work?

Answer 7

The sequence is programmed on a computer and the first nucleotide is fixed to a support such as a bead, the step remaining nucleotides are then added until an oligonucleotide is produced (about 20 nucleotides in length).

Question 8

Where does in vivo cloning take place?

Answer 8

Inside an organism.

Question 9

Give two examples of vectors.

Answer 9

Plasmids or Bacteriophages.

Question 10

Describe in vivo cloning.

Answer 10

The vector is cut open using same restriction endonuclease as was used to separate the DNA fragment. The vector and fragment are then mixed together with DNA ligase which joins the sticky ends of the fragment to the sticky ends of the vector.

Question 11

What is recombinant DNA?

Answer 11

The combination of the vector DNA and the fragment DNA.

Question 12

What does DNA ligase do?

Answer 12

Join the ends of the sticky DNA fragment and the vector

Question 13

How is a plasmid inserted into a bacteria cell/

Answer 13

Soaked in cold calcium chloride to make the cell walls more permeable, when the plasmids are added the bacteria are heated to 42 degrees for 2 minutes.

Question 14

What are marker genes used for in in vivo cloning?

Answer 14

Inserted into the plasmid along with the DNA fragment so the bacteria that have taken up the recombinant DNA can be identified.

Question 15

Give an example of marker genes used in in vivo cloning.

Answer 15

One for antibacterial resistance, so only those who have taken up the recombinant DNA will survive when grown on ampicillin.
UV light, those which have taken up recombinant DNA will fluoresce.

Question 16

What must be included if the recombinant DNA is to produce a protein?

Answer 16

Specific promoter and terminator regions.

Question 17

What do promoter and terminator regions do?

Answer 17

Tell DNA polymerase when to start transcribing and when to stop.

Question 18

Where does in vitro cloning/amplification occur?

Answer 18

Outside of a living organism

Question 19

What process is used for in vitro amplification?

Answer 19

Polymerase chain reaction (PCR)

Question 20

Describe PCR.

Answer 20

Reaction mixture must contain free nucleotides, primers and DNA polymerase. The mixture is heated to 95 degrees to break H bonds. Its then cooled to between 50 and 65 degrees to the primers can anneal. Its then heated again to 72 degrees so DNA polymerase can work and allow formation of new complementary strands.

Question 21

What are primers?

Answer 21

Short pieces of DNA that are complementary to the bases at the start of the fragment you want.

Question 22

Give three areas in which recombinant DNA technology can benefit humans.

Answer 22

Agriculture, Industry and Medicine.

Question 23

What are the two types of gene therapy?

Answer 23

Somatic and germ line.

Question 24

What is gene therapy?

Answer 24

Altering the defective genes inside cells to treat genetic disorders and cancer.

Question 25

What gene therapy is used if the disease is caused by two recessive alleles?

Answer 25

Add a working dominant allele.

Question 26

What gene therapy is used if the disease is caused by a mutated dominant allele?

Answer 26

Silence thee dominant allele by sticking a it of DNA in the middle so it cannot be transcribed any more.

Question 27

What can be used to locate specific alleles of genes?

Answer 27

Gene probe.

Question 28

What is a gene probe?

Answer 28

A short strand of DNA that is complementary to the base sequence of a target allele. It will hybridise to the target allele. Usually has a label attached so it can be detected.

Question 29

What are the most common forms of label for a DNA probe?

Answer 29

Fluorescent or radioactive

Question 30

How is allele identification using DNA probes carried out?

Answer 30

Sample of DNA is digested using restriction enzymes then separated by gel electrophoresis. The separated fragments are then transferred onto a nylon membrane and incubated with the probe. If the allele is present the DNA probe will hybridise.

Question 31

How is a DNA microarray used?

Answer 31

A microarray is a glass slide with spots of DNA probes attached. Labelled DNA is washed over the slide, if the DNA is complementary to any probes it will attach. The array is then washed and visualised under UV light of X-ray film.

Question 32

What uses are there for screening using DNA probes.

Answer 32

Identifying inherited conditions, determining how patients respond to specific drugs, help identify health risks associated with the inheritance of specific alleles

Question 33

What is genetic counselling used for?

Answer 33

Advising patients and their relatives about the risks of genetic disorders and their treatments.

Question 34

What is personalised medicine?

Answer 34

Tailored to an individuals DNA to predict how you will respond to certain treatments.

Question 35

What does VNTR stand for?

Answer 35

Variable number tandem repeats.

Question 36

What are VNTRs used for?

Answer 36

Everyone has unique introns, a combination of those from both parents.

Question 37

How is a genetic fingerprint made?

Answer 37

PCR is used to make many copies of the areas of DNA containing VNTRs. A fluorescent tag is added and then the fragments undergo gel electrophoresis. The bands produced are the genetic fingerprint.

Question 38

What can genetic fingerprinting be used for?

Answer 38

Determining genetic relationships and determining genetic variability within a population.