6.3 Manipulating Genomes Flashcards
DNA sequencing
technique that allows you to read the sequence of bases in dna
First method of dna sequencing
invented by Fred Sanger = Sanger method / chain termination methd
Explain the Sanger method
- He would get the dna double strand and turn it into a single strand
- He would allow the strands to be broken into diff bits of diff lengths
- He had 4 beakers, in each beaker he added that mixture
- In the first beaker he’d add the letter A but a modified version (radioactive dna) that whenever it’s incorporated the chain would stop and not extend further (second had modified c, 3rd = modified t, 4th = modified g)
- In the beaker there’s also normal nucleotides, dna polymerase and primers (short sequences of dna that act as the starting bit for dna polymerase to work = dna polymerase will start working to form complementary strands, each time a base would be added the chain would be stopped, thousands of chains of diff lengths and if u order them in terms of shortest to longest length you would get the sequence)
How to order the chains from the Sanger method
Use gel electrophoresis to find out shortest to longest
Small chains = further away after a set amount of time because longer chains would take longer to travel
Benefit of modified bases in Sanger method
modified in a way that they’re radioactively labelled which means during the gel electrophoresis process they’re easier to see.
How do they get lots of strands of the template for the Sanger method
- They get multiple strands of the template through PCR (way to amplify dna = polymerase chain reaction) or get your dna and put it into the plasmid of a bacteria = reproduce rlly fast
Can you sequence a whole genome
NO it would take too long
High throughput sequencing (HTS)
umbrella term for sequencing being automated + faster now
Example of HTS method
Pyrosequencing
What’s pyrosequencing
instead of using radioactively labelled dna, they took away the radioactivity = no longer radioactive but it was able to generate light so each letter basically had a diff light colour = each letter had a diff light intensity
How to work out which base is which in pyrosequencing
use mass spec to get peaks instead. = first peak was x length which shows its this base
What was pyrosequencing used for
Sequencing the human genome
Human genome project
diff scientists form diff parts of world sequenced genome + combined it all together
Why was the human genome project important
can now do genome wide comparisons between diff organisms = shows evolutionary relationships.
The human genome conserved genes
most conserved genes must have played an important role in the development of an organism
Methylation
our dnas can be methylated in different ways which can affect how 2 organisms are even though are genes are the same = affects how genes are turned on or off
What is methylation an example of
Epigenetics
What’s Epigenetics
factors that affect your dna without affecting your base sequence
What can you predict through genome sequencing
amino acid sequence of proteins
Synthetic biology
use your biological knowledge to create something artificial
Applications of synthetic biology
making proteins, biosensors, nanotechnology, medications
Bioethics
ethical issues of using synthetic biology
DNA profiling
- using dna you can create a profile and use this for CSI (forensics, paternity tests etc)
- Using dna technology to check each person has a specific profile
How to see banding pattern in dna profiling
- Through gel electrophoresis you can get a banding pattern of your dna which will be different to others
VNTRs?
Variable number of tandem repeats
What are VNTRs
sequences of dnas that are used in dna profiling. Every person has a different number of tandem repeats
What are tandem repeats
repetitive segments of dna that don’t code for a protein. They’re usually 10-100 base pair long
Outliner dna profiling
- 1 Obtain dna from mouth swabs, blood, hair, bone remains
- 2 Digest it w restriction enzymes = if you’re comparing 2 peoples dna you must ensure the same restriction enzyme is used
- 3 load it onto the gel electrophoresis
- 4 the smaller the fragment, the further it’ll travel
- 5 a banding pattern is produced
- 6 compare the banding pattern e.g. for paternity test the banding pattern of the child should be half of the mum’s and half of the dad’s
PCR?
Polymerase chain reaction
What’s pcr used for?
Used to amplify dna
What’s needed for PCR
Double stranded DNA - to act as a template
Free nucleotides (A,G,C,T)
DNA primers - signals to Taq polymerase where to bind and start synthesising
Taq polymerase - form of DNA polymerase (catalyses formation of H bonds between bases)
Buffer - maintains pH
Where does pcr ocur
In a thermocycler
Where’s taq polymerase found
Extracted from thermophilic bacteria
What is thermophilic bacteria
Can withstand high temperature without denaturing
Steps of pcr
Denaturing, annealing, elongation
Denaturing
happens at around 94-96 degrees
Denaturing happens so hydrogen bonds between complementary bases break.
What do you start pcr.
your dna you want to replicate and nucleotides and primers and dna taq polymerase + it’s co factor magnesium ions
Annealing
mixture cooled down to 68 degrees for this to happen. Primer binds to strand
Elongation
mixture heated to 72 degrees. DNA polymerase works forming phosphodiester bonds between nucleotides. ALWAYS 5’ TO 3’
What happens after the 3 steps of PCR
- Whole process repeats itself for many cycles to create more. Exponential growth (1 to 2, 2 to 4, 4 to 8)
Applications of PCR
- research e.g. if you wanna find a mutation
- Covid tests = identifying viral infection + perform as many tests as you want on the copies
What does electrophoresis do
- separate proteins or dna based on their sizes
Explain set up of gel electrophoresis
- You have a gel and make wells in it at the top and load it up with DNA.
- Put a power supply in the tank with a cathode on the side near the wells and the anode on the other side (dna is negative so it’ll move towards the anode)