DNA Techniques Flashcards
(41 cards)
How does gel electrophoresis work
DNA migrates to the positive pole in electric field.
DNA charge is proportional to length so proportional to mass
Smaller molecules move faster through the gel = separation
What 2 fluorescent dyes are generally used with DNA
Intercalating agents - go between base pairs in double helix
Minor groove binding - molecules attach to grooves on outside
What colour light would you use to excite DNA binding dyes
Blue light
What colour light would you use to detect emission from DNA
Orange light
What makes the dyes fluorescent
One or more electrons can absorb light - causes excitation - jumps to a higher energy orbital and emits light as it falls
What is a polymerase chain reaction
Rapidly makes millions to billions of copies of a specific DNA sample, allows scientists to amplify small DNA to study it in detail
What factors had been discovered to make PCR possible
- first DNA sequencing method had been developed
- synthesis of oligonucleotides was possible
- supply purified DNA polymerase to synthesis in vitro
- several DNA polymerase were isolated
Describe the steps in the PCR cycle
- Mix DNA with nucleotide triphosphates and primers
- Increase temp to 92 to denature double helix
- Lower temp to 45-65 degrees for primers to anneal to add reverse complementary bases/sequence
- Polymerisation starts
- Increase temperature for optimum DNA polymerase
- DNA is duplicated from the RNA
- Denature and anneal again to keep creating copies
Advantages of the PCR
Sensitive - works well with little initial material
Specific - will exclusively copy the target sequence
Fast - each cycle takes 2 mins, 30 cycle ~2 hours
Disadvantages of the PCR
Knowledge limited - can’t apply to an unknown DNA molecule
Length limited - limited to smaller molecules
Hot - due to denaturing
Imprecise - limited to precision of polymerase alone
How do you copy RNA
Reverse transcriptase
What was the problem with trying to copy Viral RNA
Viruses don’t have a double helix DNA genome, just single stranded RNA.
Needed reverse complement sequence to copy genome template to make copies of genome.
What did scientists do to be able to copy RNA
- Purified a viral protein that can synthesis DNA using RNA as a template
- Incubated in vitro the purified viral genome and purified viral protein
- Added tritiated deoxytymidine triphosphate (dttp) - only used to make DNA
Made tritium into a polymer - made of DNA
How is complementary DNA made
- Use oligo made of polytheoxytimidine - bind to polyA tails and synthesise reverse complement of RNA
- RNA degrades
- Single strand DNA used to synthesise can be used to synthesise the DNA strand that’ll be a copy of mRNA
- Double stranded DNA is made from mRNA
What is QRT-PCR
Quantitative reverse transcriptase - polymerase chain reaction.
Amplifies gene of interest from copy DNA
How does quantitative reverse transcriptase - PCR work
- Fluorescent dye is added to minor groove
- Measures the amount of double stranded DNA at the end of each extension step
- Done in a thermal cycler and spectrophotometer
- Plot a graph - show increase in double stranded DNA conc
How do you read a graph of QRT-PCR
The higher concentration of the target sequence = the earlier the curve goes up
Aspects of short tandem repeats
- simple short sequence repeats
- 6nts
- number of repeats is variable between individuals - valuable genetic markers
- strand slippage = reason for variability
How does strand slippage occur
- DNA polymerase synthesises repetitive DNA
- New strand detaches from template strand and is behind original position
- Results in copying one or more repeat twice = misalignment
- New strand is a different length to the template
Give 2 forensic uses of microsatellites
High polymorphism
DNA fingerprinting
For what reason might you want to build a genetic profile
Crime cases with ancestry.
Identifiying mass casualties after an explosion or natural disaster
Identify a species for food safety
How do you visualise DNA with PCR
- Use primers flanking the microsatellite
- Run fragments in a gel
- Repeat with multiple microsatellites for richer band patterns
Other uses of PCR
Genetic testing and data analysis
Ancient DNA
Creatures trapped in permafrost
Studies on evolution and geology
What did scientists have to do to work out the function of 5’ UTR and 3’ UTR
- isolate specific DNA segments
- obtain the sequence
- analyse the function
