Chapter 5: Exploring Genes and Genomes Flashcards
Acts as precise scissors for cutting specific DNA sequences
Restriction enzymes
Allows the separation and identification of specific proteins and nucleic acids using gel electrophoresis
Blotting techniques
The genome sequences of entire organisms can be determined
DNA sequencing
Specific sequences of nucleic acids can be synthesized and used to identify or amplify other nucleic acids
Solid-phase synthesis of nucleic acids
Allows the billion- fold amplification of DNA to obtain sufficient quantities for further characterization
Polymerase chain reaction
Identifying unknown restriction fragments on a gel?
Identifiying an DNA sequence on a gel, using a phosphate labeled probe.
Southern blotting
Identifying an RNA sequence on a gel using a complementary radio labeled DNA probe is called …?
Northern blotting
Using a radio labeled antibody to identify a protein on a SDS-page gel is called
Western blotting
What is needed for PCR
1) Pairs of primers
2) All four DNTPs- A,C,T,G
3) A heat stable DNA polymerase
4) Thermal cycler (machine that cycles between different temperatures.
What is the process of PCR cycle
1) Strand separation- 2 strand of the target DNA molecule separated by heat 95c
2) Hybridization of primers- solution is cooled to 54c to allow the primers to hybridize to the 5’ and 3’ ends of the target DNA
3) DNA synthesis - solution is heated to 72c which is optimal temperature for DNA synthesis by Taq DNA polymerase.
What is PCR used for?
1) Medical Diagnostics- HIV detect early stage
2) Forensics- DNA amplified Crime identificant
3) Molecular archaeology- evolutionary study
Recombinant DNA technology
new combinations of DNA sequences, which can be inserted into various organisms
DNA ligase
Requires ?
joins DNA fragments together
- a free 3’- hydroxyl group and a 5’- phosphoryl group
- Both DNA must be double helical
- An energy source such as ATP for joining DNAs
Plasmids
circular DNA molecules which can accept novel DNA sequences
Phages
viruses that infect bacteria
Mutagenesis
making mutations in genes to stud function or to gain new functions
Cloning
DNA ligase can be used to insert novel DNA sequences into a DNA vector
- commonly used vectors are plasmids and phages
How are vectors prepared for cloning ?
by cutting with a suitable restriction enzyme followed by ligation with target DNA
What are the two kinds of vectors used for cloning
Plasmids and Phage
What are plasmids?
circular double stranded DNA molecules that occur naturally in some bacteria.
What are Phages ?
are viruses that infect bacterial cells and replicate
What are the two modes of infection for a Lambda phage
- Lytic pathway
- viral functions are fully expressed
- leads to destruction of the host cell and release of hundred of virus particles
Lysogenic pathway
- the phage DNA is integrated into the host genome and can be replicated together with the host DNA
What are the pros of Lambda Phage as a Cloning Vector ?
- Phages can tolerate larger DNA insertions than plasmids
- These modified viruses enter bacteria much more easily than plasmid vectors.
What are the three kinds of mutations can be made?
1) Deletions
2) Substitutions
3) Insertions
A specific sequence within a larger DNA can be excised using ___________
The remaining ends are joined together by _____________
Can use __________ to make targeted deletions of any size —> overlap extension ______
Restriction enzyme
DNA ligase
PCR
How is mutant protein made by substitution?
it can be made containing a single amino acid substitution using oligonucleotides (primers) with the desired mutation.
mutant primer is annealed to the DNA template and is elongated using DNA polymerase.
Insertion method?
involves cutting plasmid DNA with two different restriction enzyme to remove a specific region, a new synthesized DNA fragment containing the compatible ends is then ligated into the plasmid and it allows the swapping of one gene for another.
What is the process for cloning ?
DNA ligase is used to insert DNA into a DNA vector, the vector is inserted into the host.
The vectors are prepared by cutting it with a restriction enzyme followed by ligation with a target DNA (must have compatible ends)
How are Plasmids used for cloning?
They carry genes for a selectable marker (antibiotic resistance), Contain a site that tolerates insertion of a new DNA sequence.
What are the two selectable markers for pUC18
- Ampicillin resistance- selects for bacterial cells containing the plasmid
- Beta- galactosidase- allows for blue/white color selection to determine which bacterial cells contain the DNA insert
What are Phages ?
What are the two modes of infection of a Lambda phage ?
also called bacteriophages, phages are viral that infect bacterial cells and replicate
- Lytic pathway: viral functions are fully expressed
- leads to destruction of the host cell and release of hundreds of virus particles - Lysogenic pathway: the phage DNA is integrated into the host genome and can be replicated together with the host DNA.
Why is Lambda phage better used as a vector compared to a plasmid ?
- can tolerate larger DNA insertions than plasmids
- These modified viruses enter bacteria much more easily than plasmid vectors.
What is a Genomic library?
- look over the creation of Genomic library
It contains a large number of phage particles containing fragments with the entire genome.
