Producing DNA fragments Flashcards
Why do we need to produce DNA fragments?
To isolate the gene of interest (the one you want to clone)
What are the three methods we use to create DNA fragments?
Restriction Endonucleases
Reverse Transcription
Gene Machine
Describe the process of making DNA fragments via the process of Reverse Transcription:
A cell that naturally prodcues the protein of interest is selected (e.g. pancreas cells for insulin)
These cells should have large amounts of mRNA for that protein
The reverse transcriptase then joins the DNA nucleotides with complementary mRNA bases
Single stranded cDNA is made (complementary DNA)
Then to make the DNA double stranded, you add more DNA nucleotides in and DNA polymerase.
What is the advantage of creating DNA fragments via reverse transcription?
The cDNA is intron free as it was made from an mRNA template strand (which has no introns)
Describe the process of producing DNA fragments using restriction endonucleases:
Restriction endonucleases are enzymes that cut up DNA
They have active sites which are complementary to specific DNA base sequences - described as recognition sequences
Each enzyme cuts the DNA at a specific site
When some enzymes cut - they create ‘blunt ends’ (straight down) when others cut they create ‘staggered ends’ and leave ‘exposed DNA bases’
The staggered ends are palindromic and are known as ‘sticky ends’ as they have the ability to join to DNA with complementary base pairs.
What does palindromic mean?
Same sequence forwards and backwards
‘racecar’
How do you make DNA fragments using the Gene Machine?
Scientists examine the proteins of interest and determine the amino acid sequence, then the DNA/mRNA sequence
They enter the DNA sequence into the machine, checks for biosafety/security
It creates small sections of overlapping nucleotides that make up the gene
These oligonucleotides can then be joined to create the DNA for the entire gene
PCR can be used to amplify the quantity and make it double stranded
What are the benefits of the gene machine?
Quick, accurate and makes intron-free DNA so can be transcribed in prokaryotic cells
