Topic 8B: DNA Fragments Flashcards
What is recombinant DNA technology?
transferring a fragment of DNA from one organism to another.
Why can we transfer DNA fragments from one organism to another?
Because our genetic code is universal
Because transcription and translation mechanisms are pretty similar
What are organisms called when they have transferred DNA in them?
Transgenic organisms
What are the three was DNA fragments can be isolated?
- Using Reverse Transcriptase
- Using Restriction Endonuclease
- Using a Gene Machine
Why are DNA fragments isolated?
Because in order to transfer a gene you have to first have the DNA fragments which contains that target gene.
Outline the steps of using reverse transcriptase
- many mRNA molecules are complementary to target gene
- the complementary mRNA is mixed with free DNA nucleotides and reverse transciptase enzyme
- The enzyme is used to make cDNA
Example of reverse transciptase being used
Insuline is made in the pancreatic cells. You need lots of this so but only have two copies so reverse transciptase is used to make cDNA from insulin mRNA
What is a palindromic sequence?
A sequence consisting of antiparrallel base pairs base pairs read in opposite directions)
Outline steps of using restrcition endonuclease
- the restriction endonuclease enzyme recognises the specific palindromic sequence and cut the DNA here.
- This is called a recognition sequence
- the shape of the cut recognition sequence is complementary to the enzyme’s active site
- between the recognition sequence could be the DNA and you use the enzyme to isolate the DNA fragment
- restriction endonucleas cuts DNA fragment vis HYDROLSIS reaction
Using restriction endonucleas can produce…
sticky ends and straight ends
Outline steps of using a gene machine
- the sequence that is required is designed
- first nucleotide is attached to it using a bead for support
- Add more nucleotides in order
- Add a protecting group
- Once complete the protecting group i s removed
- the oligonucleotide is joined to others to make longer DNA fragments
What is a protecting group?
Its group that makes sure that the nucleotides are joined at the right points to prevent unwanted branching
What is short sections of DNA called?
Oligonucleotides- roughly around 20 aa long
What do you do after isolating DNA fragments?
You amplify it- make it into may copies.
Identify two ways you can amplify DNA fragments
- in vivo (inside living organisms)
- in vitro (outside iving organismsl)
Step 1 of in vivo cloning
- Vector DNA is cut open using the same restriction endonuclease enzyme
- the sticky ends of the vector are complementary to the sticky ends of the DNA fragments containing genes.
- vector DNA and DNA fragment are mixed together
- they combime together with DNA ligase
- this is called LIGATION
- recombinant DNA formed
Step 2 of in vivo cloning
-Recombinant DNA is transferred into host cells
What is a vector?
something that transfers DNA into the cell eg plasmid or baceriophage
Differences between the plasmid vector and bacteriophage
-plasmid vector is persuaded into host cells where as the bacteriaphage’s DNA is injected into the cell
What is the environment in bacterial host cell?
- placed in an ice cold calcium chloride
- makes cells more permeable
- plasmids added to mixture and heat shocked (around 42 degrees around 1/2 minutes)
- this encourages the cells to take in the plasmids
How do you identify transformed host cells?
by adding marker genes in at the same time as vectors.
Name types of marker genes?
Fluorescence- through UV light you can see them
Antibiotic Resistance- transformed cells will survive and grow
Why is it important to identify transformed host cells?
Because only around 5% of host cells are taken up by DNA. Identified transformed cells can be grown and cloned more
To produce proteins you need…
promoter and terminator regions
What are promoter regions?
DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA
What are terminator regions?
DNA sequences that tell the enzyme RNA polymerase to stop
Where can promoter and terminator regions be found?
in vectors or can be added along with fragments
What is in vitro cloning
Amplifying DNA fragments outside living organisms by using polymerase chain reaction
