Topic 8- producing DNA fragments Flashcards
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
What is the second step of the gene machine?
-DNA sequence typed into the computer
-Checks that the protein being made will not be a hazard to human safety (biosafety + security)- ensures it is safe and ethical
What is the 3rd step of the gene machine
-Computer creates small sections of overlapping single strands
-These are called oligonucleotides ( a type of gene)
What is the 4th step of the gene machine?
-Oligonucleotides are joined to create the DNA for the entire gene
-1 copy
What is the 5th step of the gene machine?
-PCR can be used to amplify the quantity to make double stranded DNA
What is the advantage of the gene machine?
-Very quick + accurate
-Intron free so can be transcribed into prokaryotic cells
What is the disadvantage of the gene machine?
-Have to know the amino acid sequence which can be time consuming
Define recombinant DNA
The transfer of fragments of DNA from one organism, or species, to another
the genetic code is 1.___, as are transcription and translation mechanisms; the transferred DNA can be 2.____ within cells of the recipient (transgenic) organism
1.universal
2. translated
