DNA 2- Cloning and engineering Flashcards
What can’t sequencing tell you
Can’t tell you the function of the section of DNA
Go on to use other techniques e.g DNA cloning
DNA cloning in Bacteria
-Screen the host for certain phenotypic ( the morphology- how the phenotype looks )features
Screen the bacteria for changes in these after the insertion of our DNA
What needs to be the case for double stranded DNA to be stably maintained within host bacteria
They have to be circular
- After restriction digestion you have linear DNA
- These are difficult to maintain in bacteria
- First need to circularise it
How do you circularise double stranded DNA
One way to do this is to put this foreign DNA into a plasmid
Chop the plasmid up by restriction digestion and then add foreign DNA (that is already restriction digested)
Then stitch back together through ligation
The two pieces of DNA will stick to each other and re circularise (Now called a recombined plasmid)
This is reintroduced into bacteria (this reintroduction is call transformation )
As bacteria rapidly divide they produce many copies of the original DNA
Once grown, we can reextract the plasmids from the bacteria.
Therefor making many copies of our recombinant plasmids
What is a plasmid
Small circular piece of DNA
It is extra chromosomal- Not integrated into the genome of the organism
Often contains genes that are beneficial for the host at that particular stressful condition.
Plasmid can replicate independently of the main genome
Where do we get plasmids from
Extract these from bacteria
What is the process of ligation
Combines pieces of DNA together
Carried out by one enzyme called DNA ligase
Three Key steps in cloning
(particularly in bacteria
Restriction digestion
Ligation
Transformation
How can we use these mechanisms
To understand the human genome more
Pre sequencing this was how we understood the human genome.
Split up the human genome
and attach each nucleotide to a plasmid –> insert each one into a bacteria and see how they display themselves
How big is the human genome
3 Billion base pairs long
Genomic library
Bacteria with single pieces of a foreign organism all together form this
Can be used to discover
- Where a gene of interest is in a genome
- What are the genes that are surrounding it
- What do the control elements look like
- study the context of the gene
Colonise derived from a single bacterium isolated on a petri dish will represent each human clone
Once you have genomic libraries you can get back the plasmids run them on a gel and figure out the context of the gene of interest
How do you find out which section of DNA contains the gene of interest
Hybridisation
Steps of hybridisation
- Restriction digest your DNA
- Run that by gel electrophoresis
- Transfer the seperated fragments onto a nitrocellulous membrane
- Then carryout the hybridisation process
1. Heat up the membrane so that the double stranded DNA will now be single stranded
2. Whilst the membrane is still hot, add the probe. ( a single stranded molecule that contains all the nucleotides that are complimentary to the DNA sequence of interest)
3. Once this oligonucleotide probe is added, reduce the temp. The probe will then bind to the fragment of DNA with the complementary sequence
If we don’t know what the genome sequence is, how can we manufacture the probe for the gene we want
- Look at the protein sequence
- Proteins and genes can be sequenced in different ways
- Proteins can be sequenced using mass spectrometry
- Using the genetic code you can convert the protein sequence into an mRNA sequence and further into a DNA sequence ( this works because we know what the amino acid codons are)–> However there is a problem here caused by proton degeneracy.
Degeneracy built into the genetic code
Each amino acid is coded by more than one codon
What does degeneracy mean we have to do when using probes
A few different probes will have to be tested for each protein
By controlling the temperature that is used for annealing the probe with the DNA sequence of interest we can control the specificity of annealing
Using several probes is called degenerate probes
What is cDNA
Complementary DNA
What is the difference between cDNA and genomic libraries
The starting materials used for making these libraries
Genomic library - starting material is human DNA - section of the chromosome
cDNA library - Starting material is mRNA –> only exons present
- All mRNA have a poly A tail which provides a primer so we can artificially synthesise a stretch of poly T s which then hybridise to the poly a tail
If you then add reverse transcriptase ( virus that carries RNA as its genetic complement)
How do cDNA libraries get their samples
-The DNA is derived from mRNA
- mRNA is obtained from cells in culture or tissue
- Using reverse transcriptase
- Viruses that carry RNA as their genetic complement encode reverse transcriptase
- We can use it in an in vitro reaction in order to synthesise a complementary strand of DNA ( called complementary because the sequence is complementary to that of the mRNA)
- Treat with alkali to degrade the RNA
- Add another poly A primer and add DNA polymerase to get double stranded DNA
- This DNA can then be used to make recombinant plasmids–>make recombinant clones which carry the double stranded DNA –> Liperase that carries these clones which harbour only the complementary DNA from the host cells are called cDNA liperase.
What determines the temperature at which two strands will come together and anneal
For two single stranded DNA molecules the temperature that we expose them to will determine when annealing occurs is determined by the degree of complementarity between the bases in the two strands
Annealing
What two strands of DNA come together –> Hydrogen bonds are formed –> Double stranded DNA formed as a result
Highly complementary sequences anneal at what temperatures
Higher
During duplication how do new genes arise
Through Duplication
- This means most genes have close relatives elsewhere in the genome
PCR
Polymerase chain reaction
What happens during PCR
We take the entire genome (extract it and put into a tube) with a buffer
With the temperature we can control when two strands of DNA split apart and when they come together again
Add primer pairs –> These are complementary to the gene we are looking for
Heat to 95 degrees to separate the strands
Then cool down to around 60 degrees and then the primers anneal spontaneously forming hydrogen bonds –> complimentality
Increase the temp back up to 72 degrees–> this is the optimum temperature for the polymerase to be activate at –> it was isolated from a thermophillic orgasmism
- This DNA polymerase it uses the individual nuclotides to start priming –> Strand extension ( complementary strands)
This means we go from 1 double strand to two double strand
This is repeated over and over again, doubling each time
Common applications of PCR
DNA cloning
Detection of viral infections –> in the lab with cell lines –> detect viruses in the labs
In hospitals –> HIV
DNA fingerprinting
Rare proteins can be made in large amounts using cloned DNA
- It is possible to make completely new DNA molecules
- Custom DNA sequences are commonly used to synthesise very rare cellular proteins
- Only common proteins can be isolated from cells
- DNA can be ligated into an expression vector
Expression vector
Contains regulatory and promotor sequences
Recombinant protein
These are easily purified from the cell lysate –> Recombinant protein
Loss of function mutation/ Gene Knock out
Knock out genes within organisms. Replace and active DNA sequence with a DNA sequence that has been altered so protein no longer forms. You can then screen the host organism to find out how the bacteria behave when it looses that particular gene.
These can be used to reveal things about the function of a gene
Site directive mutogenesis
You can individually change amino acids and then look at which domains of the proteins are effected and you can study this
Looking at eukaryotic cells–> making recombinants
Still start with making a recombinant plasmid. The host is no longer a bacterial cell instead it is an embryonic stem cell.
an altered version of a gene is introduces into an embryonic stem cell. This is then incorporated by homologous recombination in some cells.
The rare cells and identified and expanded –> injected into an early mouse embryo. The mice are then bred.
