Unit 6.3- Manipulating Genomes Flashcards
Fred Sanger’s DNA sequencing method:
- Modified bases added to DNA to stop synthesis of DNA after each new length. Radioactive bases
- Electropheresis to be sorted by length
- Nucleotide base at end of each fragment is read according to radioactive label
- All the bases show the order of the DNA sequence
What is the modern type of DNA sequencing called?
Pyrosequencing
Pyrosequencing method:
- DNA cut into fragments of 300-800 using a nebuliser
- Degraded into ssDNA. These are the templates and are immobilised
- Sequencing primer added
- DNA incubated with DNA polymerase, ATP sulfurylase, luciferase, APS and luciferin
- An activated nucleotide is incorporated into the complementary strand
- This causes the PPi to be released
- In the presence of APS, ATP sulfurylase converts PPi into ATP
- In the presence of ATP, luciferase converts luciferin to oxyluciferin.
- This generates visible light which can be detected by a camera
- The amount of light generated is proportional to the amount of ATP available, showing how many of that base
Applications of gene sequencing:
-Human genome project
-Comparisons between species
-Evolutionary relationships
-Variation between individuals
-Predicting amino acid sequences of proteins
Synthetic biology:
-DNA computing
-Nanotechnology
-Biosensors
-Production of medicines
What kinds of DNA are used in DNA profiling?
Short tandem repeats (STR)
13 are used
What process is used for DNA profiling?
Electropheresis
What are the three stages in the PCR?
- Denaturation
- Annealing
- Elongation
What happens in the denaturation (1) phase in PCR?
- DNA mixed with Taq DNA polymerase, nucleotides, primers and magnesium ions
- Heated to 95C to break hydrogen bonds and make ssDNA
What happens in the annealing (2) phase of PCR?
- Mixture colled to 68C
- Primers anneal
- Taq DNA polymerase enzymes bind to the end with the primer
- 72C is the optimum temperature for this enzyme
What happens in the elongation (3) phase of PCR?
- Temperature raised to 72C
- Taq DNA polymerase catalyses the addition of DNA nucleotides inteh 5’-3’ direction
- Whole process repeats
- Produces lots of copies of the DNA
What does the polymerase chain reaction (PCR) do?
Amplifies short lengths of DNA to thousands of millions of copies
What does electropheresis do?
Separates DNA or proteins into fragments of different sizes
What mediums does electropheresis happen in?
Agarose gel plate covered by a buffer solution
Gel electropheresis process:
- DNA samples cut with restriction enzymes at specific sites (35-40C)
- Agarose gel poured into central region of tank; combs placed at ends. Buffer solution added to cover gel and end sections of tank
- Comb removed, leaving wells
- Loading dye added to DNA
- DNA put in wells
- Connected to battery, 6-8 hours
- Smaller fragments travel further
- Buffer solution poured away and dye added to gel. The dye adheres to the DNA and stains the fragments
Why does DNA travel towards the anode (positive electrode) in electropheresis?
Negatively charged because of phosphate groups
