DNA fragments

Explain how complementary DNA is made using reverse transcriptase

1. isolation of the DNA fragments that have the gene for the desired protein

2. insertion of the DNA fragment into a vector (bacteria)

3. Transformation, the transfer of DNA into suitable host cells

4. identification of host cells that have successfully taken up the gene by using gene markers

5. Growth/cloning of population of host cells

Explain how complementary DNA is made using reverse transciptase

beta cells specialised in insulin production make a lot of mRNA that codes insulin

mRNA acts as a template on which a single-stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase

single stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme

double stranded DNA is formed on the template of the cDNA using DNA polymerase

Explain how restriction endonucleases are used to cut DNA into fragments

Bacteria often defend themselves from viral infections by cutting up viral DNA

DNA is cut into recognition sequences. A cut is made between two opposite pairs leaves 'blunt ends'

others cut in a staggered fashion to expose a DNA palindrome and create sticky ends.

Explain the 'Gene machine'

the amino acid sequence of a protein is determined, from which the mRNA codons and DNA triplets can subsequently be worked out

the computer designs a series of small, overlapping single strands of nucleotides and oligonucleotides

a double stranded copy of this gene is generated and using sticky ends is inserted into a plasmid vector

Genes are checked using sequencing techniques and those with errors are rejected

List the gene machine advantages

- any sequence can be produced

- very short process

- very accurate

- intron free

- prokaryotes can be used