Recombinant DNA Technology

What does recombinant DNA technology involve?

Transfer of DNA fragments from one organism/ species to another, involving a number of processes. Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient organism

What are the 5 processes involved?

• Isolation/production of DNA fragments that have the gene for the desired protein

• Insertion if the DNA fragments into a vector

• Transfer of DNA into suitable host cell

• Use of marker genes to detect genetically modified cells or organisms

• Growth/ cloning of population of host cells

What are the 3 ways to isolate DNA fragments?

• Using reverse transcriptase

• Using restriction endonucleases

• Using the gene machine

What is the process of using reverse transcriptase?

• Isolate mRNA from a cell that readily synthesis the protein coded for by the desired gene (mRNA easily extracted as these cells have large quantities)

• Mix mRNA with DNA nucleotides and reverse transcriptase (the reverse transcriptase uses mRNA as a template to synthesis a single strand of complementary DNA)

• DNA polymerase is used to build up the complementary nucleotides on the cDNA template, forming a second strand of DNA. This double strand of DNA is the required gene

What is the process of using restriction endonuclease?

• Restriction endonuclease enzymes cut DNA at the specific base ‘recognition sequence' either side of the desired gene. The shape of the recognition side is complementary to the active site of the enzyme

• Many enzymes cut in a staggered fashion, forming complementary sticky ends

Why are sticky ends called sticky ends?

Their exposed bases readily form hydrogen bonds with the complementary bases in the sticky ends of other molecules, cut by the same restriction endonuclease

How can fragments of DNA be extracted using a gene machine?

• Synthesises fragments of DNA quickly and accurately from scratch, without needing a DNA template (amino acid sequence of the protein is determined, allowing the base sequence to be established)

• These do not contain introns so scan be transcribed and translated by prokaryotes

• This is a slow and expensive process, so isn’t used

What are the advantages of obtaining genes from mRA rather than directly from the DNA removed from cells?

• Much more mRNA than DNA in cells making the protein than DNA, so more easily extracted

• In mRNA, introns have been removed by splicing (in eukaryotes) whereas DNA contains introns, so can be transcribed and translated by prokaryotes who cant remove introns by splicing

How can fragments of DNA be amplified in vivo?

Adding of promoter and terminator regions

• this happens at the end of isolating your DNA fragment

• once you have obtained your required DNA fragment, we need to attach a promoter region and terminator region

• The promoter region is added so the DNA fragment ca be transcribed onto an mRNA strand in order to make the protein, so it starts the process, by allowing RNA polymerase to bind to DNA

• The terminator region is added to stop transcription at the appropriate point

What is the role of inserting DNA fragments into a vector?