Polymerase Chain Reaction Flashcards
What fo you need for DNA synthesis in vitro?
- DNA polymerase
- dNTPs
- Template DNA
- Primer
What does PCR stand for?
Polymerase chain reaction
What fo we need for PCR?
- Templete DNA
- Primers
- dNTPs
- Buffer
- Tag polymerase
- Heat
How discovered PCR?
Mullis 1993
How many cycles in the process of PCR?
30-40 cycles
What are the steps in PCR?
- Denaturation
- Annealing
- Extension
Describe the denaturing step in PCR:
- Double DNA stran melts open
- Heating sample to 95C
Describe the annealing step in PCR:
-Primers bind to DNA and polymerase attaches and starts copying DNA
Describe the extension step in PCR:
72C optimum temperature for polymerase and extension of fragments
What is PCR preformed in?
Thermocyler
What happens each time the PCR is cycles?
More strands
What is the application of PCR described as?
Exponential
Which part of the DNA is amplified in PCR?
Between primers
Can specific sequences be amplified from complex mixture of DNA?
Yes
What are the ends of the amplified fragments defined by?
2 primers
What are PCR primers?
short -20 base pairs single stranded DNA (oligonucleotides)
Are PCR primers synthesis by naturally or commercially?
Commercially
How much DNA do you need to have to visualise on an agarose gel?
1 microgram
How do we analyse DNA?
With agarose gel electrophoresis
Describe the separation of DNA by size:
- Potential difference applied along gel
- DNA moves to positive electrode through gel depending on conformation (shape)/size (smaller fragments faster than large)
- Stain DNA with fluorescent dye for detection by UV exposure
What is the processes called where PCR products being directly sequences?
Sanger Sequencing
What are the application of PCR?
- DNA sequencing
- Detection of pathogens in water
- Genetic fingerprinting
- Forensic analysis
- Diagnosis of genetic disorders
- Prenatal diagnosis
- Analysis of ancient DNA
What are the limitations of PCR?
- Sequence information is required to design 2 primers
- Limit on length of amplified fragment
- Potentially high error rate
- Very sensitive to exact reaction conditions
- any contaminating DNA will be amplified
Do bacteria have plasmids?
Yes
What is the shape of bacterial chromosome?
Circular
What are plasmids?
small extrachromosomal circles of DNA
What can DNA be cut by?
Restriction endonucleases
What can join DNA?
DNA ligase forms phosphodiester bond (requires ATP)
What is recombinant DNA?
Allows two DNA molecules to be joined
What is good about able being able to join DNA?
Genes can be inserted into plasmids
What are the overlap-based methods of DNA assembly?
PCR-based methods
- Site-specific recombination methods
- DNA repair-based methods
- In vivo homologous recombination
Describe the gene cloning:
- Introduce recombinant plasmid into bacterial cell
- Replicated
- Cell divides
- Clone of cells
- Recover DNA for analysis
- Gene cloning
What is another way you can clone genes?
Inserting gene into bacteriophage DNA vectors
What is a gene library?
- Collection of DNA put into plasmids and multiplied through transformation in a bacteria
- Collection of Recombinant clones
What is DNA hybridisation?
screening for clones containing gene of interest
What is meant by transgenic?
- Genes between species
- Generic code is universal
How can DNA be expressed in cells?
- Introduction of DNA into cell
- Expression vector inserted withe gene of DNA
- Can express gene in bacteria by inserting into a plasmid
What is in an expression vector?
a particular type of plasmid that has a promoter telling a bacteria to transcribe the gene
What has to happen before DNA can be expressed in cells?
Due to introns in mRNA is needed
-Reverse transcribed into cDNA before cloned into vector
What are the applications of genetic engineering?
- Human insulin
- Blood clotting factor VIII
- Human growth hormone
- Bovine chymosin
- Hepatitis B vaccine
- Artemisinin
What is SRY gene?
Sex determination gene
Give an example of genetic engineering?
- SRY genes make xx mice phenotypically male
- Inserting XY into XX cause male characteristics
What is a reporter gene?
Proteins tagged with green fluorescent protein
What is synthetic biology?
design of new biological parts, device, and systems and re-design existing, natural biological systems for useful purpose
What are the applications of synthetic biology?
-Artemisinin production (didn’t have to wait for crop production which effect the economical price)
