Recombinant DNA technology

Question 1

Definition of recombinant DNA

Answer 1

DNA from two different sources joined together.

Question 2

Definition of in vivo.

Answer 2

Experiments performed within the living organism.

Question 3

Definition of in vitro.

Answer 3

Experiments performed in test tubes / petri dishes outside of the living organisms.

Question 4

Describe an overview of recombinant DNA technology (10 points).

Answer 4

1. The required DNA gene is obtained from donor DNA.
2. First located with a gene probe.
3. Plasmid DNA is cut with a restriction enzyme at restriction site.
4. Donor DNA is cut with the same restriction enzyme as used to cut the plasmid.
5. The enzyme produces complementary sticky ends.
6. Donor DNA is joined into a vector, for example; plasmid using ligase enzyme- seals sugar-phosphate backbone.
7. A recombinant plasmid is formed.
8. Vector (recombinant plasmid) carries the gene into recipient cell, for example:bacterial cell, which divids the vector inside.
9. The new cells alll have the recombinant plasmid, they are transgenic.
10. When proteinsynthesis occurs, the donor gene is expressed and the required product is made. `

Question 5

What are the three methods to isolate a gene?

Answer 5

1. Conversion of mRNA to cDNA, uing reverse transcriptase.
2. Using restriction enzymes to cut a fragment containing the desired gene from DNA.
3. Creating the gene in a ‘gene machine.’

Question 6

Describe the method to convert mRNA to cDNA, using reverse transcriptase (8 points).

Answer 6

1. B-cells from the Islets of Langerhans in the human pancreas- where the insulin gene is transcribed into mRNA (no introns).
2. mRNA coding for insulin is extracted from the cell.
3. mRNA coding for insulin (no introns).
4. mRNA acts as a template on which a cDNA strand is formed using reverse transcriptase.
5. Single-stranded cDNA (complementary DNA as no introns).
6. New nucleotides form hydrogen bonds with complementary base pairs. Adenine-thymine, cytosine-guanine.
7. DNA polymerase joins the new adjacent nucleotides / forms phosphodiester bonds and double stranded cDNA is formed.
8. Copy of human insulin gene can now be inserted into host cell / cloned.

Question 7

What are the advantages of using reverse transriptase? (2 points).

Answer 7

1. mRNA is easy to obtain as it can be found outside the nucleus.
2. Using mRNA isolated from cytoplasm means introns have been removed.

Question 8

Definition of a restriction enzyme / endonuclease.

Answer 8

Enzymes which cut DNA at specific base sequences (recognition sequences).

Question 9

Describe the method for using restriction enzymes to cut the desired gene from DNA (8 points).

Answer 9

1. Restriction endonucleases are enzymes which cut (hydrolyse) DNA at specific base sequences (recognition sites).
2. Different restriction endonucleases cut DNA at different specific sequence of bases because the shape of the recognition site is complementary to the enzyme’s active site.
3. These recognition sites occur in the DNA of all species of organisms- but not in the same places.
4. These recognition sequences are palindromic- base pair can be read in opposite directions.
5. If recognition sequences for these enzymes are present at either side of the DNA fragment desired, the restriction endonucleases to separate it from the rest of the DNA can be used. The DNA sample is incubated with the specific restriction endonuclease, which cut DNA fragment out of DNA.
6. It is important that the recognition sequence for the selected restriction endonuclease does not occur within the gene wanting to be ioslated.
7. Restriction enzymes are made by the bacteria to cut out the viral DNA sequences from the bacterial DNA.
8. The cut made at the recognition site is either straight (giving a bulnt end) or staggered (producing a sticky end).

Question 10

Definition of recognition site.

Answer 10

4-8 bases long nucleotide sequence, where restriction enzyme attaches and cuts.

Question 11

Definition of palindromic.

Answer 11

Base sequence reads the same forwards as it does backwards.

Question 12

Definition of sticky end.

Answer 12

Recognition site cuts leaving staggered exposed bases. Allows / attached to complementary exposed bases.

Question 13

Definition of blunt sticky end.

Answer 13

Recognition site cuts in a straight line down.

Question 14

Describe how to create a gene in a ‘gene machine’ (5 points).

Answer 14

1. Protein sequence is analysed.
2. Amino acid sequence is worked out.
3. mRNA sequence worked out.
4. DNA nucleotide base sequence is uploaded into the computer.
5. Computer produces DNA base sequence by producing overlapping single strands of nucleotides called oligonucleotides which ca then be assembled into a gene.

Question 15

What are the advantages of using the gene machine? (3 points).

Answer 15

1. Faster to use the gene machine rather than using reverse transcriptase or restriction endonucleases.
2. There are more steps involved in isolating the gene / mRNA as well as the enzyme catalysed reactions.
3. Highly accurate.

Question 16

Describe RNA polymerase.

Answer 16

Enzyme that joins RNA nucleotides to form mRNA during transcription.

Question 17

Describe transcription factors.

Answer 17

Binds to RNA polymerase and promotor to begin transcription.

Question 18

Describe transcription region.

Answer 18

Region of DNA that releases RNA polymerase to stop transcription.

Question 19

Definition of a vector.

Answer 19

A DNA carrier used to transfer DNA into cells, for example: a bacterial plasmid or virus.

Question 20

Describe the method for how the gene of interest is put into a vector (3 points).

Answer 20

1. The vector has to be opened by using the same restriction enzyme so the sticky ends are complementary.
2. DNA ligase is then used to recombine the 2 peices of DNA.
3. This is known as recombinant DNA.

Question 21

Describe the chemical transformation that takes place to transform the DNA into the host (3 points).

Answer 21

1. Ca^2+ has a positive charge.
2. DNA has a negative charge.
3. Positive charge is attracted to negative charge so DNA will not be repelled by membrane.

Question 22

Describe how the recombinant DNA is transformed into the host cell by using heat shock (3 points).

Answer 22

1. Drop the temperature so there is less kinetic energy.
2. Quickly increase the temperature, to achieve a burst of kinetic energy.
3. Creates gaps in the membrane for DNA to move into the bacterial cell.

Question 23

How is the correct bacteria identified and what happens to the useless bacteria? (4 points).

Answer 23

1. Some of thr plasmids will have resealed without becoming recombinant.
2. Some of the gene of interest will have resealed without becoming recombinant.
3. There will be some recombinant DNA.
4. Some of the bacteria will not have transformed at all.

Question 24

Definition of antibiotic resistance.

Answer 24

The ability for micro-organsims to withstand the adverse effects of antibiotics.

Question 25

Definition of genetic markers.

Answer 25

A gene or DNA sequence with a known location on a chromosome that can be used for identification. Can be modified to be fluorescent or radioactive.

Question 26

Describe the method for how to identify transformed bacteria by replica plating (7 points).

Answer 26

1. On the master plate, all the bacteria colonies are grown.
2. The colonies on the master plate are transferred onto a plate containing antibiotics. This is called replica plating. (The colonies must be transferred in exactly the same position).
3. The first replica plate medium contains the antibiotic ampicillin.
4. The replica plate is incubated.
5. The surviving colonies are transferred onto a second replica plate.
6. The second replica plate medium contains the antiobiotic tetracycline.
7. The replica plate is incubated.