Producing DNA Fragments

Question 1

What is recombinant DNA

Answer 1

The DNA of two different organisms that have been combined by isolating genes, cloning them and transfer them into microorganisms. These microorganisms are then grown to provide a factory for the continuous production of desired proteins

Question 2

What’s the resulting organism of recombinant DNA called

Answer 2

Transgenic or generically modified organism

Question 3

What are the 5 stages of making a protein using DNA technology of gene transfer and cloning

Answer 3

1. Isolation - of the DNA fragments that have the gene for the desired protein
2. Insertion - of the DNA fragment into a vector
3. Transformation - that is, the transfer of DNA into suitable host cells
4. Identification- of the host cells that have successfully taken up the gene by use of gene markers
5. Growth/cloning - of the population of host cells

Question 4

What are the methods toproduce DNA fragments

Answer 4

Conversion of mRNA using reverse transcriptase

Using restriction endonucleases to cut fragments containing the desired gene from DNA

Creating the gene in a gene machine, usually based on a known protein structure

Question 5

How in a host cell are they able to synthesise DNA from their RNA

Answer 5

Using an enzyme called reverse transcriptase

Question 6

Describe how reverse transcriptase is used to isolate a gene

Answer 6

1. A cell that readily produces the protein is selected
2. These cells have large quantities or relevant mRNA, which is therefore more easily extracted
3. Reverse transcriptase is then used to make DNA from RNA. This DNA is known as complimentary DNA (cDNA) because it is made up of nucleotides that are complimentary to the mRNA
4. To make the other strand of DNA, the enzyme DNA polymerase, is used to build up the complementary nucleotide me on the cDNA template. This double strand of DNA is the required gene

Question 7

What enzyme defends bacteria from viruses by producing enzymes that fit up the viral DNA

Answer 7

Restriction endonucleases

Question 8

Where do restriction endonucleases cut

Answer 8

At a DNA double strand at a specific sequence of bases called a recognition sequence

Question 9

How are genes manufactured in a laboratory

Answer 9

1. The sequence that is required is designed
2. The first nucleotide in the sequence is fixed to some sort of support (a bead)
3. Nucleotides are added step by step in the correct order
4. Add protecting groups - make sure that the nucleotides are joined at the right points, to prevent unwanted branching
5. Short sections of DNA called oligonucleotides are produced.
6. Once they are complete, they are broken off the support and all protecting groups are removed.
7. The oligonucleotides can then be joined together to make longer dna fragments

Question 10

What are the advantages of the gene machine process

Answer 10

Any sequence of nucleotides can be produced in a very short time and with great accuracy

Free of introns, and other non coding DNA, so can be transcribed and translated by prokaryotic cells

Question 11

How can DNA fragments be made using restriction endonuclease enzymes

Answer 11

1. Some sections of DNA have palindromic sequences of nucleotides - consist of antiparallel bass pairs
2. Restriction endonucleases are enzymes that recognise specific palindromic sequences ( recognition sequences) and cut the DNA at these places
3. ( different restriction endonucleases cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to the enzymes active site
4. Can then separate recognition sequences at either side of the DNA fragment you want using restriction endonucleases
5. The DNA sample is incubated with the specific restriction endonuclease, which cuts the DNA fragment out via a hydrolysis reaction
6. The cut can leave sticky ends - they can be used to bind the DNA fragment to another piece of DNA that has sticky ends with complementary sequences