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DNA ligase is able to join both sticky- and blunt-end fragments together by reconstituting...
a phosphodiester bond
Describe the PCR process
- Stage 1:
- 1. DNA sequence to be amplified is identified
- 2. DNA strands are separated by gentle heating from 94-96 degrees Celsius to break H bonds and breaking apart the double stranded DNA. This differs from DNA replication as DNA helicase and gyrase breaks H bonds
- 3. DNA forward and reverse primers [3' to 5'] to sepaarte DNA strands and build complementary strands
- 4. Temperature lowered to 50-65 Celsius for the primers to anneal to the template strand.
- 5) After annealed, temperature raises to 72 Celsius so Tag polymerase can build complementary strands using free nucleotides. This differs from DNA replication bc Taq is used as it can withstand high temperatures since it derives from Thermus aquaticus. Complementary strands are build exponentially
- 6) Amplified DNA is run on polyacrylamide gel
- 7) Gel stained using ethidium bromide and viewed under UV light
After first cycle, variable length strands are prodocued that start at the target region on one end (where the primer is annealed) and extended beyond the target region. In the second cycle, DNA strands are heated and separated, primers allowed to anneal. On two of the DNA strands, one end terminates at target region, and primers anneal to the other end of target area. Taq starts adding the appropriate nucleotidesd, and ceases when the end of target region at the constant length strands
What does PCR do?
Direct method of making many copies of DNA, indirect method of cloning.
Before PCR, it was not possible to make numerous copies of a desired gene fragment unless it was inserted into a plasmid. PCR is a direct method of making copies of a desired DNA sequence.
What is PCR used for?
Criminal investigation, medical diagnosis, evolutionary relationships
What is polymorphism?
Any difference in DNA sequence that can be detected between individuals.
Organisms from the same species will have the same gene but different alleles coding for that
How does polymorphism work for coding and noncoding regions?
- Coding: detect mutations
- Noncoding: variation in VNTR
Describe the process of RFLP
RFLP - a technique in which DNA regions are digested using restriction endonuclease(s) are subjected to radioactive complementary DNA probes to compare the differences in DNA fragment lengths between individuals
restriction endonucleases -> radioactive
- 1) DNA extracted from sample is digested and run through gel
- 2) Smears gel. The gel is then submersed into a chemical to break the double stranded chromosomes to single stranded
- 3) Single stranded DNA migrates from gel to nylon membrane using an electric current (Southern blotting). This is done by placing the nylon membrane with positive electrode behind it. The DNA will transfer out of gel and onto nylon and bind.
- 4) Nylon membrane immersed in solution with radioactive complementary nucleotide probes for specific chosen regions looked for, such as mutations. Radioactive proves hybridize to complementary sequences
- 5) Nylon membrane pressed against xray film. The radioactive probe causes the hybridized areas to be exposed and makes a pattern
Compare RFLP and PCR using the following categories: state of sample, size of sample, time, basic premise, result medium, tools
- State of sample: large
- Size of sample: whole genome
- Time: three weeks
- Basic premise: cleaving DNA using restriction endonucleases followed by subjection to radiocactive complementary DNA probes
- result medium: autoradiogram
- Tools: restriction endonucleases, radioactive proves, nylon membrane, x-ray film, gel electrophoresis
- State of sample: minute [one cell]
- Size of sample: target sequence
- Time: one day
- Basic premise: building complementary strands using principles of DNA replication
- result medium: gel
- Tools: DNA polymerase, nucleotides (A,G,C,T), DNA primers, gel electrophoresis
How long does RFLP take?
What does RFLP find [sample]
What does PCR find [sample]
What is restriction endonucleases?
Bacterial enzyme that cleaves DNA sequences at specific recognition site
What are methylases?
Bacterial enzymes that add a methyl group to recognition sites to protect DNA from cleavage by restriction enzyme
What is DNA ligase?
Enzyme that joins complementary fragments by reconstituting PHOSPHODIESTER BOND of DNA backbone
What is gel electrophoresis?
Process by which DNA fragments of different lengths are separated by electrical current
What are plasmids?
small circular DNA that has the ability to enter and replicate in bacterial cells and, therefore, can be used as a vector to introduce new genes into a bacterial cell
What is the Sanger dideoxy method?
A method of DNA sequencing based on DNA replication that uses dideoxy nucleoside triphosphates
Describe the Sanger method
The method is similar to DNA replication
Goal of Sanger method is to have DNA template is treated to become single stranded in order to be sequenced
- 1) Obtain single stranded DNA and make copies
- 2) Add radioactive labelled primer to the end of the DNA template
- 3) Divide copies amongst the four test tubes that contain DNA polymerase, free nucleotides (dNTPs, and a radioactively labelled dideoxy analogue of one the the dNTPs in LOW concentration
- 4) Use gel electrophoresis to separate strands in all four tubes
- 5) Create autoradiograph
In the Sanger method, where do you add radioactive labelled primer?
End of DNA template
Why are the dideoxy analogues in low concentration?
High concentration will cause premature chain termination
What are ddNTPs?
ddNTPs are their respective dNTP whose deoxyribose sugar is missing the -OH group on C3.
Usually DNA polymerase will catalyze the phosphodiester linkage b/w 3'-OH group and the phosphate group of the incoming nucleotide, but if it isn't there, the complementary strand ends
Name three fields that genetic engineering can be applied to, and name some subheaders
- MEDICAL FIELD
- i) Screenings
- ii) Gene therapy - modify gene expression by inserting normal gene or a gene for a desired product
- AGRICULTURAL FIELD
- i) transgenic plants - foreign DNA to improve
- ii) potential problems
What did Stanley Cohen and Herbert Boyen devise?
Stanley found plasmids, Herbert found restriction endonucleases
Devised a technique of isolating, recombining, and introducing new genes into bacteria using plasmid vectors
What is Boyen's first biotech company called?
How does one create the genetically modified version of somatotropib (HGH), somatropin?
HGH has 217 AA, 26 becing AA signals. These 26 are removed, leavign only the 191 somatotropin. These are inserted in a plasmid after lac promotor and introduced in E. coli cells like in transformation
When do you use CaCl in transformation in transformation and why?
If a bacterium readily takes foreign DNA, it is a competent cell. This is not occurring naturally but can be chemically induced with CaCl.
Bacterial membrane has negatively charged phosphates. The positive charged calcium ions stabilize the negatively charged phosphates, and the low temperature makes it rigid, making the entire cell membrane stablized. Now that it's stablized, the plasmid DNA can be introduced.
What is transformation?
Introduction of foreign DNA in bacteria
Why do plasmids need an antibiotic resistance?
Plasmids must have an antibiotic resistance gene so that if you successfully transformed your bacteria, it will grow in that culture.
Describe the process of transformation
- Competent cell will go through transformation through either CaCl solution to stabilize it, electroporators (chambers that subject bacteria to electric shock to loosen cell walls and allow foreign DNA to enter), and gene guns (shoots DNA through cell wall)
- Plasmid DNA introduced in solution
- Solution subjected to heat shock at 42 degrees to create a draft so the outside enviornment had a higher temp than inside of cell
- This draft sweeps plasmids in bacterial cell thru pores of membrane
- Bacteria is incubated
Ways to input plasmid in cell in tranformation?
- CaCl solution to stabilize it
- Electroporators (chambers that subject bacteria to electric shock to loosen cell walls and allow foreign DNA to enter)
- Gene guns (shoots DNA through cell wall by droplet of water, which is charged andfired down a tube via an electrical discharge. The water droplets hit a carrier sheet thatcontains the DNA wrapped around gold particles. The force of the impact causes theDNA to be propelled toward the target cells, where it is able to penetrate cell walls andcell membranes)
Ways to clone?
- Insert recombinant DNA into bacteria, using
- a plasmid vector, to allow reproduction of bacteria and then separate the cloned
- genes from the reproduced bacteria
Major cloning steps
- 1) Generation of DNA fragments
- using restriction endonucleases to excise
- gene fragment
- 2) Construction of recombinant molecule by ligated it to
3) Introduction to host
- 4) Select – growth in colonies is
What role do methylases have in bacteria and eukaryotes?
- Bacteria, immune
- Eukaryotes, role in transcription
Which DNA ligase is more frequently used?
Describe the process of gel electrophoresis
- Power turn on, DNA moves through gel
- Loading dye
What is selective plating?
Growing a bacteria on a medium that supports the transformed cells that have foreign DNA
What types of bonds do restriction endonucleases break?
Phosphodiester bonds b/w guanin and adenine, followed by H bonds of complementary base pairs
What enzymes produce sticky ends?
EcoRi, Sall, HindIII
What enzymes produce blunt ends?
How do you read a Sanger dideoxy graph?