21.1 Recombinant DNA technology - Producing DNA fragments

Question 1

What does recombinant DNA allow

Answer 1

It allows genes to be manipulated, altered and transferred from organism to organism - also DNA to be transferred

Question 2

What is recombinant DNA

Answer 2

When the DNA of two organisms is combined

Question 3

What is the result of recombinant DNA technology called

Answer 3

A genetically modified organism

Question 4

Why is the DNA of organisms accepted and functions normally when its transferred into a different species

Answer 4

Because the genetic code is the same in all organisms and is universal

Question 5

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

Answer 5

1. Isolation
2. Insertion
3. Transformation
4. Identification
5. Growth/Cloning

Question 6

Breakdown of DNA technology stages

Answer 6

Isolation - of the DNA fragments that has the gene for the desired protein

Insertion - of the DNA fragments into a vector

Transformation - The transfer of DNA into suitable host cells

Identification - Of the host cells that have successfully taken up the gene by use of gene mutations

Growth/cloning - Of the population of host cells

Question 7

What are the 3 methods producing DNA fragments

Answer 7

1. Using reverse transcriptase for conversion of mRNa to rDNA
2. Using restriction endonucleases to cut fragments that contain the desired gene from DNA
3. Creating the gene machine that usually requires a known protein structure

Question 8

How is reverse transcriptase used to produce a DNA fragment

Answer 8

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

Question 9

How are restriction endonucleases used to produce a DNA fragment

Answer 9

1. Each restriction endonuclease cuts a DNA double strand at a specific base sequence, called a recognition sequence.
2. Sometimes this occurs between two opposite base pairs. This leaves blunt ends
3. Other restriction endonucleases cut in a staggered fashion, leaving staggered ends

Question 10

How is the gene machine used to produce DNA fragments

Answer 10

1. The desired sequence of nucleotide bases of a gene is determined from the desired protein that we wish to produce
2. The amino acid sequence of the protein is determined and from this the mRNA codons are worked out and the complementary DNA triplets are worked out
3. The desired sequence of nucleotides is entered into a computer
4. The computer designs a series of small, overlapping single nucleotide strands called oligonucleotides. These can be assembled into the desired gene
5. In an automated process, each of the oligonucleotides is assembled by adding one nucleotide at a time in a required sequence.
6. The oligonucleotides are then joined to form a gene. These gene has no introns and is replicated by the polymerase chain reaction
7. The polymerase chain reaction also constructs the complementary strand of nucleotides to make the required double gene, it also multiplies the gene to give numerous copies
8. Using sticky ends, the gene can be inserted into a plasmid. This acts as a vector, allowing it to be cloned.