Topic 8- producing DNA fragments Flashcards
(32 cards)
What is the 1st in recombinant DNA?
-To produce or isolate fragments of DNA so they can be combined with another peice of DNA
What are the 3 methods used to produce DNA fragments?
-Restriction endonucleases
-Reverse transcriptase
-Gene machine
What is the function of the reverse transcriptase?
-Makes DNA copies from mRNA
Where is reverse transcriptase naturally occurring?
-In viruses (such as HIV)
What is the first step in reverse transcriptase?
-A cell that naturally produces the protein of interest is selected
-These cells produce large amounts of mrna (for that protein)
-mRNA is extracted
What is the process that reverse transcriptase carries out called?
-Reverse transcription
What is the 2nd step in reverse transcription?
-Enzyme reverse transcriptase joins DNA nucletides together
-Which are complementary bases to the mRNA sequence
What is the 3rd step in reverse transcriptase?
-Joined nucleotides create single stranded DNA, cDNA (complementary DNA)
What is the 4th step in reverse transcriptase?
-If it is needed to be double stranded
-More nucletides are added
-DNA polymerase
What are the advnatges of reverse transcriptase?
-Introns are alreday removed (as it is based on the mRNA template), this is espically important for prokaryotic cells as they cannot remove them
-Lots of copies of mRNA are alreday present so they are avaliable for cDNA
What are the disadvantages of reverse transcriptase?
-More steps in comparison to others
-More time consuming
-More technically difficult
What is the function of restriction endonucleases?
-Enzymes that cut DNA
Where are restriction endonucleases naturally occurring?
-In bacteria
-As a defence mechanism (so foreign DNA cannot replicate and harm the bacteria
How are restriction enzymes complementary to a range of different DNA sequences?
-They all have a slightly different active site
Where is the DNA cut (as it is cut at a specific location)?
-Wherever the RE is complementary to a range of different DNA sequences
How is a blunt end formed?
-A vertical cut down the DNA molecule
Can some enzymes cut at the same location on the double DNA strand
Yes
How is a staggered end (sticky end) created?
-Cut at palindromic sequences so the end is staggered, leaving exposed DNA bases
Why are sticky ends the most useful?
-They have the ability to join the DNA with complementary base pairs
What is meant by palindromic?
-Bases read the same way forwards as they do backwards
What is the advantage of using REs?
-Sticky ends make it easier to insert so easier to make recombinat DNA
What is the disadvantage of the REs?
-Still contain introns as it cuts the original DNA (which is an issue of bacteria)
What is the gene machine?
-Carried out in a lab
-Using a computerised machine
What is the 1st step of the gene machine?
-Scienetists examine the protein of intreast to identify the amino acids sequence (primary strucrture)
-Work out what the mRNA (codeded for by AAs) and DNA sequence would be
