Chapter 5

What are restriction enzymes? What are they also known as?
These are enzymes that recognize certain base sequences in dsDNA and cleave them. They can be used to sequence and create new DNA molecules. Also known as restriction endonucleases.
What do prokaryotes use RE for?How long are the cleavage sites?
Used to cleave foreign DNA. Their own DNA is methylated at cleavage sites. 4-8 BP.
What is a unique characteristic in the method the RE uses when cleaving sites?
There is a twofold axis of symmetry. For example the sequence that is read from 5'-3' should be the same when read from 3' to 5'.
What is an isoschizomer?
Restriction enzymes with similar cleavage sites.
What are the three ways the RE can cleave certain DNA sites?
5' overhang, 3' overhang, or blunt ends
-leaves a PPi on the 5' and a hydroxyl group on the 3' end of both strands
What are some of the methods used to ISOLATE nucleic acids? To SEPARATE?
-Some of these methods include precipitation of proteins.
Describe the first type of electrophoresis used to separate nucleic acids.
-Pulse field gel electrophoresis uses a pulsing electric field to stretch and relax large DNA molecules from different directions. The fragments are then seen with autoradiography or stained with ethidium bromide (orange glow).
Describe the second type of electrophoresis used to separate nucleic acids.
This would be called SOUTHERN blotting. It separates the fragments according to size, denatured, then rejoined with the complimentary strand probe (P32) to identify the exact fragments.
What is Southern, Northern, and Western blots?
Southern is blotting with DNA, Northern is blotting with RNA, and Western is blotting with protiens (and ABs).
Describe the Sager sequencing method of DNA.
1) DNA is denatured into a ss (template strand).
2.) DNA is placed into 4 testubes along with a primer, polymerase, and dNTPs.
3.) each testtube will contained a differently modified ddNTPs.
4.) Electrophoresis will be used to identify the complimentary sequence.
Describe the Florescence sequencing method of DNA.
1) DNA is denatured into ss. 2.)Primer is added with plymerase.
3) Complimentary strand is synthesized suing dNTPs and terminated with labeled ddNTPs so it terminates at every single nucleotide in different strands.
4) Placed in gel electrophoresis that can measure the different lengths of DNA.
5.) Using a florescence it can determined the sequence of DNA.
What are the three methods used to clone DNA?
Plasmids, bacteriophages, and PCR.
What are plasmids and how are they purfied?
Plasmids are small circular DNA components of bacteria that can replicate independently.
1) Bacteria is broken up and the DNA is isolated
2) after isolation the DNA is denatured (both plasmid and chromosomal).
3) The DNA is then renatured and normally the chromosomal DNA has a harder time renaturing but the plasmid DNA renatures very well.
4)The two are separated using high-speed centrifugation.
What are some important qualities for a plasmid to have when using it as a DNA vector?
-easy to replicate
-genome should be well known
-contain a selectable marker to be identified
-contain an insertional inactivation
-ampicillin resistant
Describe the method used to replicate DNA with plasmids.
1) restriction enzymes cleave segments out of the plasmid
2) the desired segment to be replicated is added using DNA ligase.
3) the Ecoli cells are treated with calcium to make them permeable to DNA.
4) the Bacteria are placed in agar and treated with ampicillin, so only select cells can replicate
5) The cells replicate and create sister copies of plasmids.
What are the commonly used phage vectors?
Commonly used phage vectors are M13 and gamma.
Describe the method used to replicate DNA with bacteriophages.
1) restriction enzymes cleave out the area to be removed.
2) DNA ligase is used to attach the foreign DNA
3) virus infects bacteria and uses host machinery to replicate; integrates into bacteria DNA.
4) the newly synthesized DNA, head, and tail of virus combine and lyse the cell releasing thousands of newly replicated foreign DNA.
What is used as a replication promotor region in bacteria plasmids?
What is a YAC?
This stands for yeast artificial chromosome. This is when LARGE DNA fragments can be inserted into the yeast and subsequently cloned.
Steps in PCR (polymerase chain reaction).
1) strand separation 95 C
2) hybridization of primers on 3' of both strands
3) heated for DNA synthesis. Synthesis extends beyond target sequence. Polymerase that is used is heat stable (thermus aquaticus)
4) by 3rd cycle, short strands exponetialy grow while the long strands arithmetically grow.
What are some applications of PCR?
-Detect infectious agents (HIV)
-Prenatal dx of genetic diseases
-Genetic fingerprinting
-amplification of rare DNA
What is the polymorphism analysis for determining the presence of huntington disease?
This is done by searching for the segments of DNA that have the mutation. If there is no mutation there is no cleavage noted in the electrophoresis. It can also be determined if the patient is hetero or homozygous.
What is the point of electrophoretic mobility shift essay?
This allows us to identify the proteins that DNA bind to. If the movement is slow this means that the DNA is tightly bound to proteins, if not it is easily disassocated .
What is complimentary DNA and how is it synthesized?
Complimentary DNA is synthesized when mRNA is reverse transcribed into single stranded cDNA. sscDNA is then made into dscDNA with polymerase. This can be cloned using a vector.
What is the formula for determining the probability of a desired sequence? Number of fragments?
N=Log (1-P)/Log(1-f)
Describe the steps of site directed mutagenesis.
1) First heat the circular DNA and have the template strand.
2) Create a primer that is modified with a point mutation
3) Place the polymerase to replicate the DNA.
4) Heat this DNA molecule to separate it into the 2 strands.
5) Take the coding strand and use a polymerase to make it into dsDNA and you will have this point mutation.
Describe cassette mutation.
1) Plasmid DNA is cut by restriction enzymes
2) new sequence with mutation is added.
What is a designer gene?
These are genes that can be mutated to create things that do not exist in nature such as:
-immunotoxin antibodies used against cancer (Research)
-vaccines making non-infectious viral coats
-new proteins with no counterparts
Transgenic animal are animals that express a gene from another animal. What is the process that is carried out to accomplish this?
The gene of interest is injected into the pronuclei near a promotor region that is activated by certain factors. In this case the promotor metallothionein is activated by Cadmium. This metal was added to the water and this caused the tested mice to grow much larger than the control.
Describe a possible tx of SCID (Severe combined immunodeficiency) using gene therapy.
-This condition is caused when the stem cells in the bone marrow are unable to code for IL-7 which is essential to the creation of T-cells.
-Removing the marrow from the children and treating them with a vector containing the normal gene then returning the marrow
-children were able to lead normal lives
What are some short-comings of gene therapy for SCID?
-effective for a short period of time
-side effects