Creating DNA Fragments

Question 1

How is mRNA converted to double stranded cDNA?

Answer 1

1. mRNA strands are isolated
2. Reverse transcriptase and free DNA nucleotides are added to make single stranded cDNA
3. Lots of copies of single stranded cDNA are produced
4. Free DNA nucleotides and DNA Polymerase form double stranded cDNA

Question 2

How are restrictions enzymes used to cut a specific gene from a chromosome?

Answer 2

1. DNA is isolated from the nucleus using detergents to break down the cellular and nuclear membranes and histones are removed using digestive enzymes
2. Required gene is cut out from the rest of the DNA using restriction endonuclease at specific palindromic restriction sites
3. Results in an isolated gene with complementary sticky ends

Question 3

How are gene machines used to make genes?

Answer 3

1. The amino acid sequence is entered into a computer and the DNA triplets are obtained, allowing the base sequence to be determined (degenerate so may not determine the natural base sequence)
2. A gene machine links together DNA nucleotides together to make the gene with no introns present and no promotor or stop region