DNA technology Flashcards
Define genome
the length of DNA in 1 haploid set of chromosomes entire gene set
What are the 4 basic genetic engineering tools?
- Plasmid vectors
- Restriction enzymes to cut DNA
- Gel electrophoresis to separate DNA fragments
- DNA ligase to join DNA fragments
What are the 5 basic steps of gene cloning?
- The gene is isolated
- Gene is inserted into vector
- Transported into a host cell
- Multiplication of recombinant DNA
- Division of host cell colony
Why is gene cloning important?
To generate large amounts of DNA
To generate large amounts of desired protein
To detect mutations in gene sequencing
What 4 things does PCR require?
ds DNA template
Pair of oligonucleotide primers
DNA polymerase
Nucleotides
Why is Taq DNA polymerase used?
Taq DNA Polymerase has a temperature optimum of 72°C and is relatively stable at 94°C compared to early polymerases
What are the 3 steps in the PCR that require different temp and what are they?
Step 1 – Denature template DNA to ssDNA with heat (95)
Step 2 – Lower temperature to allow primers to anneal (55)
Step 3 – DNA Polymerase extends primer - Go to step 1 (72)
Every cycle of Polymerase Chain reaction the total DNA template is ……..
Doubled
What are the limitations of PCR?
: Difficult to amplify products longer than 10-15kb. : Need to know the sequence flanking your gene
What is special about cycle 35?
Increasing the cycle number above 35 has little positive effect: reagents are depleted
: polymerase is damaged
Why is less than 10,000 base pairs used?
Easily purified
Avoids DNA breakdown
What are plasmid vectors derived from?
Derived from Plasmids found in bacteria
What does Ori mean?
Ori- origin of replication
What are restriction enzymes used for?
And how do they work?
Cut DNA by hydrolysing phosphodiester bonds
as they recognise specific sequences of DNA bases
Describe Host-controlled resistance
Phage injects DNA into bacterium
Restriction enzymes bind to DNA from phage
Phage DNA is cleaved and inactivated
Why don’t restriction enzymes cleave bacterial DNA?
Contains an ECO RI methylase - Methylates the sequence GAATTC
Bacteria dont have this
DNA methylate selectively methylate’s what?
Hemi-methylated DNA
Define Exonucleases
Removing nucleotides from the ends
Define Endonucleases
Break nucleic acid chains in the interior
Explain what Type I,II and III of endonucleases do
Type I- cuts DNA 1000 bases from recognition site
Type II- cuts DNA at recognition site
Type III- cuts DNA 25 bases from recognition site
Describe the nucleophilic attack that occurs when RE break a phosphodiester bond
Arrow from Nucleophile B to H from H20
Arrow from O in H20 to P in phosphate
Arrow from P-O to H-A
Arrow from bond between H-A to A
Leaves a O- on phosphate
Describe how to do gel electrophoresis
a) Separation of DNA fragments by gel electrophoresis
b) Visualising DNA in a gel with nucleic acid binding dyes and UV light e.g. Ethidium bromide or Gelred.
Describe how a gene gets added to a plasmid// form recombinant DNA
Requires enzyme DNA ligase (Usually T4 DNA ligase)
Which catalyses a phosphodiester bond
And requires ATP
What are the two types of ligation?
Blunt end and sticky ends
True or false you can cut the vector and gene with the same and different RE
True
True or false you can use nucleases to create blunt ends
True
True or false you can use DNA pol to fill in ends
True
Name a typical linker you can use to join vector and the desired gene
BamHI site
What are the 5 options to construct a recombinant DNA molecule
- Cut vector and gene w/ same RE
- Cut with different
- If gene and vector dont have compatible enzymes use nucleases to create blunt ends and DNA pol to fill the ends
- Use linkers and adaptors
- Use TA cloning
What is TA cloning?
Adds a single 3’ adenine overhang to each end by adding thymine and terminal transferase
What is transformation?
Introduction of DNA into living cells
What are the 3 steps in transformation
- Prepare competent bacterial cells
- limited normal uptake
- chemical/physical treatment
- Introduce DNA into cells at 50mM CaCl2 treatment
- Select those cells that have taken up the recombinant DNA via 42. 2 min heat shock
Define selection
most vectors confer antibiotic resistance which helps determine which cells harbour the recombinant DNA molecule. (how we determine which have taken up the gene
Transformation is inefficient. Why?
Transformation is an inefficient process.
- Only a small % of plasmids are taken up. - Approximately 0.01% of cells take up DNA.
Define Tetr and Ampr
Ampr= Ampicillin resistance gene Tetr= tetracycline resistance gene
What are the 3 ways we can distinguish bacteria that have taken up the recombinant DNA or self ligated vector?
Replica plating (pBR322 vectors)
Blue/white selection (pUC vectors)
Restriction mapping (any vector)
Describe replica plating
What is the marker? and what is its role?
Tetracycline marker
Helps us distinguish between recombinant and self ligated vectors
Describe Blue/white selection
Which gene is used and what does it code for
Lac z gene encodes for Enzyme beta galactosidase - selectable marker
Beta galactosidase converts lactose into galactose and glucose
X-gal is an analog of lactose and turn blue when cleaved by Beta galactosidase
What are the 4 steps of restriction mapping?
Transformation ligation mix into bacteria
Plate out on agar + ampilcillin
Grow up bacteria and extract DNA
Using restriction enzymes to distinguish between recombinant and self ligated vector
Recombinant DNA has more markers
To produce protein from a cloned gene you need what?
An expression vector
What do expression vectors provide?
Provide transcription and translation.
Expression vectors.
What catalyses transcription and how?
What happens in bacteria
Transcription is catalysed by RNA polymerase
RNA pol binds to the promoter region of a gene
In bacteria - ribosome attaches to the mRNA via a ribosome binding site (rbs).
Why choose bacteria or animal cells as host cells
i) Bacteria - Grow in liquid media
- Divide every 20mins
- Bacteria do not glycosylate proteins
ii) Animal cells - Need to grow attached to a solid matrix
- Divide every 18 hours
- will glycosylate proteins
Factor Viii is produced in animal cells
What does it play a central role in?
Plays a central role in blood clotting
Commonest form of haemophilia results from inability to synthesise Factor VIII
What is a bioreactor?
Animals are bioreactors which have foreign gene inserted into them. eg spider silk in milk
