DNA Technology - Biochemistry
What is gene cloning
Where a gene is located and then is inserted into a vector to produce recombinant DNA, where then the DNA is then cloned and amplified to get a large amount of a gene type
What is a cloning vector
They are used during gene cloning in order to generate a large amount of DNA
They are derived from small circular DNA called Plasmids in bacteria and can replicate independently from the host cell
How does Gene cloning happen
S1= Isolate the desired gene from the DNA
S2= Insert the isolated DNA to the vector in order to produce recombinant DNA
S3= Transport the Vector into a host cell such as E.coli
S4= Then the recombinant DNA is inserted to host cell DNA and then udergo replication of the cell to many cells
What is an expressor vector
If you want to extract a specific protein from a gene type, where vector is imnserted to animal cell for desired protein to be produced, rather than the gene being extracted in larger quantity
Why is Gene cloning important
To generate large amounts of DNA
Generate large amount of a protein from a gene of interest with expressor vector
For Gene sequencing and expression
For the detection of disease causing mutations and dtermine chance of it happening
Develop new ways to treat disease and cure inherited diseases by gene therapy
How do you Isolate a gene
With the use of PCR:
Need to design 2 primers to anneal at either end of the gene
Need to then lyse the DNA and PCR it to get enough DNA to clone into vector
What is needed for PCR to take place
DNA polymerase in the form of TAQ polymerase due to optimum at 72 and stabel at 94 degress celcius
Pair of oligonucleotide primers
Free nucleotides and a desired DNA template
How does PCR work
S1= Denature the DNA to ssDNA by using heat to break hydrogen bonds at 94 degrees
S2= Lower temperature to 72 to then allow primer to bind vai hydrogen bonding to complimentary pairs
S3= The TAQ polymerase then add other nucleotides via complimentary base pairing to extend the primers
What are the limotation of PCR
Hard to amplify prodjucts longer than 10-15 Kbp
What is needed for the gene to be inserted to the vector
Plasmid Vector
Restriction enzymes to cut the DNA
Gel electrophoresis to seperate DNA fragments
DNA ligase to join fragements together
What are restriction enzymes that cut DNA
They recognise specific sequences of bases in the DNA and can bind to them
Then they cut the double stranded DNA by bydrolysing phosphodiester bonds
Why can't restriction enzymes cleave bacterial DNA
Due to contain ECO RI methylase that will methylate the GATTC sequence
What is DNA methylation
DNA is replicated by semi-Conservative replication
DNA methylase selectively methylates hemi-methylated DNA
What are the 2 types of restriction enzymes
Exonuclease- Remove nucleotides from the end of the DNA chain
Endonuclease- Remove nucleotides from the middle of the DNA chain
Types of restriction endonucleases
Type 1 - Cut DNA 1000BP from recognition site
Type 2 - Cut DNA at the recognition site
Type 3- Cut DNA 25 BP from the recognition site
What are blunt end and sticky ends
When they cut they make blunt ends that are harder to join together comopared to others that make sticky ends that are easier to join together as greater chance of complimentary base pair binding to take place of overlap happens
What is the recogniton site
Many restriction enzymes recognise the hexanucleotide site and other recognise 4,5 or 8 nucleotide sequences
What is Gel electrophesis
The analysis and seperation of DNA fragments made by the restriction endonuclease
The DNA loaded in wells and then move from negative to postive charge
Can see the seperated DNA in the gel under UV light
How does DNA ligase work to join DNA fragment together
Needed to produce recombinant DNA by joing 2 DNA strands together by catalysing the formation of phosphodiester bonds during a condensation reaction and ATP is needed
Can be done with sticky or blunt ended fragments but stick yhas greater chance of happening
How is Recombinant DNA made with ssame restriction endonuclease
Cut vector and gene with same enzyme, to leave the same bidning sequence at the end of the ligation
Can only happen if gene have same sequence at both end
Produce sticky ends for DNA ligase
How to make recombinant DNA with 2 restriction endonuclease
Cut Vector and gene with different enzymes, to leave differing sequences at end of the genes,
Need the vector to have both sequences in the restriction site and need to use 2 enzymes to create 2 stciky ends for DNA liagse
How to make recombinant DNA with no compatible enzymes
Use nucleases to create blund ends due to cut off njon compatible sticky end
Carry out blunt end ligation or use DNA Polymerase to fill in sticky ends rather than cut to make blunt ends
How to use linkers to make recombinant DNA
Can use linker where blunt end undergo ligation to create sticky ends bu adding linkers to them
How to use TA cloning to make recombinant DNA
Method to clone PCR products, by manipulate them to have sticky ends and then can manipilate the vector by adding small bases with terminal tranferase to make sticky ends on the vector
How to introduce the DNA to living cell
This is called transformation before the recombinant DNA can be multiplied
You need to prepare a suitable bacteria cell before can instert the plasmid
How to prepare a competent bacteria cell
Treat bacteria with calcium chloride so plasmid bind to cell exterior
Heat shock at 42 degrees to increas membrane permeability so then plasmid can enter the bacteria
Then grwo tranfored bacteria on agar plate to form colonies of it
Only 0.001 % of E.coli wil be tranformed due to most vectors die in ampicillin so grow on amipicillin playe then only the vector with the recombinant DNA will grow due to have resistance gene
How to distinguish the recombinant DNA and self ligated bacteria
Replica plating for PBR322 due to tetracycline is selectable marker in the vector
Selectable marker help to distinguish the recombinant and self ligated
Recombinant DNA make tetra inactive
What is Blue White selection
Lac z gene endode for enzyme B galactosidase that is selectable marker
Enzyme convert the lactose to galactose and glucose
If have recombinant DNA then lac z wont work so colony is white not blue so then remove all the white ones as recombinant vectors
What is restriction mapping
Transform the ligation mix into bacteria
The plate this on agar+ampicilin
Then grow the bacteria and extract the DNA and use restriction enzymes to distinguish by gel electrophoresis
What the expression factors to grow the host cell
To produce a protein from target gene need an expression vector to allow protein synthesis of wanted protein
Transcription catalysed by RNA polymerase and RNA pol bind to promoter sequenece of the gene
If need an animal protein then add bacteria promoter sequence with is LAC promoter
Why choose a bacteria host cell
Grow in liquid medium and divide rapidly
Dont glycosolayte protein so no further modification takes place
Why use animal cell as host cell
Need to grow attached to solid matrix
Allow post translational modification to allow protein to function
Slower division
Need to use animal cloning vector
How to make Bioreceptors
Use animals to produce certain benefical proteins
Gene inserted to animal and get proteins from:
Chickens via eggs and cows via milk