DNA Analysis and Technology Flashcards
What is a restriction endonuclease?
DNA endonucleases that recognize and cut specific palindromic DNA sequences in a very specific manner.
Generating staggered/sticky ends that are antiparallel and complementary to one another.
How can restriction endonucleases be used to clone?
Two DNA fragments cut with the same RE will have the same sticky ends –> hybridize them together (ligate), thus cloning two pieces of DNA together.
Ways in which recombinant DNA cloning has been used in translational medicine (producing therapeutic agents)
Clotting factors, growth factors, human insulin, vaccines, monoclonal antibodies, etc
Hybridization
Technique to determine the presence of a target sequence for isolation.
- Use a single-stranded radio-labeled DNA probe that is antiparallel and complementary to the target sequence.
- Autoradiography to detect probe
- Can compare X-ray film to original master plate to isolate colonies that contain the gene of interest
Southern blotting (hybridization technique)
- Genomic DNA Is digested with an RE
- Gel electrophoresis to separate diff size fragments
- Transfer to nitrocellulose
- Hybridize with target probe
- Autoradiogram tells us the size of the fragment that contains the target sequence
Norhtern blot
Probe the total mRNA of a cell with your gene of interest to determine how that gene is expressed.
Ex) Does expression chang eif the cell is grown under two diff conditions?
Western blotting
Determine how a target protein is expressed in a cell, using a specific antibody as a probe.
Reverse transcriptase, as used by retrovirsues
In the host, the retrovirus’s RNA genome is used as a template by reverse transcriptase to generate double-stranded cDNA that gets integrated into the host genome for replication –> viral proteins
Use of reverse transcriptase in microchip/micraoarray analysis
- mRNA is isolated from two distinct cell populations
- Reverse transcribe it into two different fluorescently labeled cDNA pools (green and red)
- Apply fluorescent pools onto a microarray of every gene in teh genome
The more intese color tells you that more of that gene is expressed in that pool.
Microarray analysis in medicine & bioinformatics
- Characterize tumor by its expression profile
- Predict better treatments by using cells grown with and w/o the drug
- Ex) If the drug kills cancer by inducing paoptosis, we should see induction of pro-apoptotic gene expression
How does the structure of dideoxy nucleotides (ddNTPs) allow for DNA sequencing?
ddNTPs have a hydrogen instead of a 3’OH group, which would terminate DNA synthesis when incorporated.
If you fluorescently label each ddNTP and mix it with DNA and all the dNTPs, then gel electrophorese it, the shortest fragment represents the first nucleotide added at the 5’ end of the fragment.
PCR
Amplification of a gene using
- Heat-stable DNA polymerases
- Antiparallel complementary sequence-specific primers that flank both ends of a sequence or gene of interest
PCR in recombinant cloning
If the primers have a unique RE site, then the amplified fragment can be digested and cloned into a vector
PCR in diagnosis & forensic analysis
Use PCR enzymes to amplify and verify a mutation to diagnose a disease state (or confirm identity in forensic analysis)
Polymorphisms
Genomic variations that naturally occur in a gene within the population (too common to just be a new mutation).
Maintained through evoluation because they confer something helpful. Ex) Resistance to malaria
