What is cloning
- Cloning involves the recombination of DNA fragments together and making copies of them
- Bacterial plasmids often recombined with new DNA
How can recombinant dna be useful
- Mutating genes to look for altered phenotype
- Introducing wildtype genes to see if it rescues a mutant
- Linking reporter genes to follow gene activity
- Can be used to work out sequence of a gene
Red, white, green, blue and yellow biotechnologies
Red: production of recombinant proteins for medical processes
White: enzymes for industrial processes
Green: nutrient supplementation/resistance for agriculture
Blue: aquatic sources of genes for biotech
Yellow: sources of genes from insects/spiders
Requirements for DNA cloning
- Enzymes for manipulating DNA
-restriction enzymes for cutting
-ligating enzymes for joining
-replication enzymes for synthesising/amplifying e.g. polymerases - A cloning vector e.g. plasmid
- A host cell to implant plasmid
- A gene of interest
How do restriction enzymes work
- Bacterial enzymes primarily used to destroy invading bodies
- Work by recognising palindromic sequences and asymmetrically cleaving the sequence either side of the symmetry axis
- Asymmetric cuts = staggered cuts = overhangs = sticky ends which can bind to complementary sticky ends (important for recombination)
- DNA ligase joins nucleotides of fragment ends by covalent bonding
What processing do plasmids require to be analysed in terms of size
- Linearising by cutting once with a restriction enzyme
Describe the steps of DNA recombination of a plasmid with a gene
- Plasmid and foreign DNA cut by the same RE
- Sticky ends of the gene anneal to sticky ends of the plasmid
- Nicks in sugar-phosphate backbone sealed by ligase
- But if sticky ends of vector anneal with itself, the lacZ gene is expressed forming an enzyme which will break down X-gal substrate, producing a blue dye
- So white bacterial colonies shows that the foreign gene has disrupted the lacZ gene and are thus the recombinants (desired)
What requirements for PCr
- Denaturation at 95 separates strands
- Forward and reverse primer anneal to template strands at 55
- At 72 extension occurs where new strand is synthesised
- Usual length of primer is 18-22 bases meaning the chance of the sequence occurring is 1/4 ^18
Why does target length DNA not appear till cycle 3 of PCR
Only at this cycle does the enzyme find the forward and reverse primers of the the region to be amplified
What are the applications of PCR
- Mutation detection
- Screening for infectious agents in cells
- Genotyping
- Forensic analysis
How to check whether the colonies of bacteria that are white truly have a the correct inserted foreign dna
- Using PCR and then gel electrophoresis will give the size of the foreign DNA
- Should match up with the ladder if present
How to control the expression of the inserted gene
Subclone the gene into an expression vector regulated by a bacterial promoter that only switches on when recognised by the required proteins to initiate transcription
Expression vector structure
- A bacterial antibiotic reststance gene
- Multiple cloning sequences are restriction enzyme sites that allow the insertion of the gene
- A promoter
- A terminator (transcription stop sequence)
- Another antibiotic resistance gene that gets switched on when the expression vector is implanted with a gene
- Can contain reporter genes
How to isolate a gene
If the sequence is known PCR can be used since we can design primers complementary to the gene
If the sequence is not known but there is a homologous gene known we can assume this homologous gene is identical e.g. isolating dog cardiac muscle but only have a human and mouse gene sequence
If the protein sequence is known, primers can be designed but due to degeneracy of the code, there can be multiple sequences per amino acid so complementarity is tricky
If the gene is short it can be auto chemically synthesised
How can isolated genes be verified
Probing against a DNA library of the same gene in other organisms
A DNA library exists as clones of plasmids that contain the genetic material of an organism
This is done to mitigate the chance of replication errors
Southern blot
Method of verifying gene presence
- Cleave dna into single stranded fragments via RE
- Run on a gel to separate fragments by size
- DNA fragments on the gel transferred/immobilised onto a nitrocellulose paper by suction (southern blot formed)
- Random labelled SS DNA ‘guesses’ are introduced which bind to the complementary sequences on the SS DNA original
- These labelled SS DNA act as primers for DNA synthesis
- Denaturation of the strands will yield one radioactive SS DNA
- This SS DNA used to probe the original southern blot and bind to the complementary sequence which will fluoresce/emit radiation
How to make a genomic DNA library
- Cleavage of DNA with RE
- Insertion of fragments into plasmids
- Recombinant plasmids introduced into bacteria
How to screen a DNA library
- Place recombinant bacteria on a nitrocellulose sheet
- Cell structure is destroyed and the residual DNA binds to nitrocellulose
- Labelled probe attaches to target sequence
- X-ray film detects probe
- Comparison of the bacterial colony with a replica shows which colony contains the target gene
Give several reasons why making a genomic library can be problematic
- A large genome may be too large to screen for a particular gene accurately and efficiently
- Some genes contain many introns and can be an issue when cloning into a plasmid
- Expression of a gene containing introns is impossible for bacteria since they cannot remove introns
Solution: cDNA, complementary DNA which is complementary to mRNA and only includes the expressed gene sequence
How to make dna from rna
Comparison of cDNA with genomic library
- Reverse transcriptase enzyme used to form cDNA from the mRNA template
- Primer bins to the mRNA Poly-a-tail and allows reverse TSCPtase enzyme to form cDNA
- Ribonuclease H degrades the mMRN allowing a complementary cDNA homologous to the mRNA strand to be formed
- cDNA is a library of functional gene sequences, minus the introns
- Screening for a gene with a labelled probe occurs similarly as in genomic libraries
When to use genomic library or cDNA
- If interested in cloning regulatory regions e.g. promoters genomic DNA is preferred since cDNA does not contain these regions (only contains the mRNA sequence in double stranded DNA form)
- If expression of gene to make protein is wanted, if levels of mRNA expression is wanted, cDNA better
How to screen expression of a protein
- Using an expression vector that makes the protein of interest
- Lyse bacteria, transfer to nitrocellulose and add a labelled antibody specific to protein
- Spot(s) on nitrocellulose identifies colony of interest
Give an instance when making cDNA the difficult way is not needed to get the gene of interest
- Using PCR when the RNA sequence is already known
- Mix with reverse transcriptase enzyme and poly dT primers
- Produces cDNA
DNA microarrays
How to use them to measure gene expression changes in a tumour
- Collection of all the genes of an organism/cell type
- Prepare cDNA from both normal cells and tumour cells using fluorescent dNTPs (two different colours)
- Add the separated strands of cDNA to a microarray allowing the fluorescent strands anneal to complementary sequences
- Spots on the microarray will be either one colour, the other colour or a mix of both, and will represent expression patterns of different genes
