Flashcards for Exam 2
starting with "recombinant DNA technology"
What is Plasmid DNA
Small circular pieces of self-replicating DNA found primarily in bacteria.
What is Recombinant Plasmid DNA
A type of plasmid DNA that has been artificially altered to include DNA from another source (such as a gene of interest).
Plasmid traits
- Considered extrachromosomal
- Approx 1 to4 kilobases.
What are vectors
Pieces of DNA that can accept, carry, and replicate other pieces of DNA
What is Calcium Chloride Transformation of Bacterial Cells
a method used to introduce foreign DNA into bacterial cells.
- “inefficient process”
Process of CaCl2 Transformation
- bacterial cells are immersed into a cold CaCl2 solution for 30 minutes.
- plasmid DNA is added, and heat ( 30 secs at 42 C) shock temporarily opens the membrane, allowing DNA to enter.
- plasmid DNA enters bacterial cells and is replicated and expressed in their genes.
What is Electroporation
applying a brief pulse of high voltage electricity to create tiny holes in the bacteria cell wall to allow DNA to enter.
What are the advantages of Electroporation
- rapid and more efficient
- require fewer cells
With reference to recombinant bacteria, what is “selection”
Selection is the process of identifying recombinant bacteria while preventing the growth of non-transformed ones (contain plasmid without foreign DNA)
What is Antibiotic Selection
a method used to identify bacteria that have successfully taken up a plasmid containing a gene for antibiotic resistance
- cannot distinguish between bacteria that have taken up the desired recombinant plasmid and bacteria that have taken up a plasmid that has recircularized without the successfully taking it in.
What is Blue-White Selection
a technique used to identify recombinant bacteria. Bacteria with plasmids containing the lacZ gene can hydrolize X-gal, turning blue. If foreign DNA is inserted into the plasmid, the lacZ gene is disrupted, and these bacteria remain white.
What and when were the first recombinant human proteins marketed
Insulin in 1982 and Growth Hormone in 1985
Technique for insulin
inserting the cloned human insulin cDNA sequence into a plasmid. The plasmid is then introduced into bacterial cells, which are used to synthesize the insulin protein encoded by the cloned gene.
What was the source of growth hormone prior to recombinant technology
human cadavers
Practical Features of DNA Cloning Vector
- Size: Small enough to be separated from the host’s chromosomal DNA.
- Origin of replication (ori): Starting point of DNA replication, allows plasmid to replicate independently.
- Copy number: plasmids per cell; usually low, but have high copy number.
- Multiple cloning site (MCS): Contains recognition sites for several restriction enzymes where DNA inserts are cloned.
- Selectable marker genes: Allow selection of transformed colonies.
- RNA polymerase promoter sequences: Enable transcription in vitro and in vivo.
Why cant you use human insulin genomic DNA for a cloning project
genomic DNA contains introns (non-coding regions), which bacteria cannot process.
What are DNA Libraries
collections of DNA fragments that represent the complete genome of an organism. “held” in plasmid vectors within host bacteria (ex. E. coli)
What are the two types of libraries
- Genomic DNA Libraries
- Complementary DNA libraries cDNA
Genomic libraries:
- Isolate chromosomal DNA from tissue of interest and digest with a restriction enzyme to produce fragments.
- Digest the vector with the same enzyme for compatible sticky ends.
- Use DNA ligase to ligate genomic DNA fragments with plasmids or adapters/linkers.
Disadvantages of Genomic Libraries
- Inclusion of Introns: Both introns and exons are cloned; most genomic DNA in eukaryotes is introns, resulting in a library rich in non-coding DNA.
- Search Difficulty: Large genomes make finding the gene of interest challenging and time-consuming.
- Gene Expression Info: Does not provide data on levels of gene expression.
Making complementary DNA from mRNA
- Extract mRNA from tissue of interest
- Use reverse transcriptase to create double-stranded DNA from mRNA.
- Add short linker DNA sequences with restriction sites to cDNA ends.
- Cut with a restriction enzyme and ligate with a cut vector to create recombinant vectors.
- Transform bacteria with recombinant vectors.
Advantages of cDNA Libraries
- Represents actively expressed genes from the tissue from which mRNA was isolated.
- Does not include introns.
- Can isolate genes expressed under specific conditions in a tissue.
Disadvantages of cDNA Libraries
Difficult to create if the source tissue lacks abundant mRNA.
Library Screening - Process of Colony Hybridization
- Grow bacteria with recombinant DNA on an agar plate.
- Place a nylon or nitrocellulose filter over the plate.
- Treat the filter with an alkaline solution to lyse bacteria and denature DNA; expose to UV light.
- Denatured DNA binds to the filter as single-stranded DNA.
- Incubate the filter with a probe tagged with a radioactive nucleotide or fluorescent dye.
- Probe binds to complementary sequences on the filter (hybridization).
- Wash the filter to remove excess unbound probe.
- Expose the filter to X-ray film or a digital camera to detect the fluorescent probe.
- Compare the film or image to the original agar plate to identify colonies with the gene of interest.
Polymerase Chain Reaction (PCR)
A technique for amplifying target DNA quickly.
Developed in 1983 by Kary Mullis.
PCR Process- What components are needed for a PCR reaction?
Target DNA, nucleotides (dATP, dCTP, dGTP, dTTP), buffer (MgCl₂), DNA polymerase, and forward and reverse primers.
PCR Process- What is the role of the buffer in a PCR reaction?
Maintaining the optimal pH and ionic strength for the DNA polymerase to function effectively.
(Typically contains MgCl2).
PCR Process- What are primers in PCR, and what is their function?
Primers are short single-stranded oligonucleotides (18-22 nucleotides long) that bind to the target DNA to guide DNA polymerase for replication.
PCR Process- How is the PCR reaction initiated after adding all components?
The reaction tube is placed in a thermocycler, which amplifies the target DNA through repeated cycles of heating and cooling. thermocycler for amplification.
PCR Cycle in Thermocylcer - What are the three stages of a PCR cycle?
- Denaturation (94-96°C)
- Annealing (52-58°C)
- Extension (70-75°C)
What happens during the denaturation stage of PCR?
DNA is heated to 94-96°C to break hydrogen bonds between the strands, separating them for replication.
What occurs during the annealing (hybridization) stage in PCR?
Primers hydrogen bond with their complementary sequences at the 3’ ends of the target DNA at 52-58°C.
What happens during the extension (elongation) stage in PCR?
DNA polymerase copies the target DNA at 70-75°C, synthesizing new DNA strands.
What is the result of one PCR cycle?
The amount of target DNA doubles at the end of each cycle.
How many times is the PCR cycle typically repeated?
The PCR cycle is repeated 20-30 times.
Advantage of PCR
- Amplifies millions of copies with a small amount of starting material in a short period of time.
-To calculate # of copies - T x 2^N
N = # of PCR Cycles
T = represents the # of initial target DNAs - quicker and more effective than DNA libraries
What DNA Polymerase is used for PCR
Taq DNA Pol - isolated from a species known as Thermus Aquaticus that thrives in hot springs.
Disadvantage of PCR
Depends on the design of the primers that match the target sequence. Poorly designed primers can lead to non-specific amplification, resulting in unwanted products.
What does Taq Polymerase do
it puts a single adenine nucleotide on the 3’ end of all PCR products
How do researchers use the adenine added by Taq polymerase in PCR products?
Researchers use a T-vector that has single-stranded thymine on each end, allowing it to base pair with the adenine in the PCR products for cloning purposes.
DNA Sequencing - What is the Sanger Method used for?
The Sanger Method, or chain termination sequencing, is used to determine the sequence of nucleotides in a cloned gene.
Who developed the Sanger Method and when?
The Sanger Method was developed by Frederick Sanger and colleagues in 1977.
What components are required for the Sanger sequencing reaction?
The reaction requires a single primer, denatured DNA template, all 4 dNTPs, DNA polymerase, buffer with MgCl₂, and dideoxynucleotides (ddNTPs).
How do dideoxynucleotides (ddNTPs) terminate DNA synthesis in the Sanger Method?
ddNTPs have a 3’ H instead of a 3’ OH, preventing the formation of a phosphodiester bond with the next nucleotide, causing chain termination.
What is the advantage of high-throughput computer-automated sequencing using the Sanger method?
- more than 600 nucleotides can be sequenced per reaction (using capillary electrophoresis)
- essential in completing the Human Genome Project
- uses only 1 reaction tube rather than 4
How are ddNTPs detected in Sanger sequencing with capillary electrophoresis?
ddNTPs are labeled with different fluorescent dyes, and a laser scans DNA fragments in a capillary tube, causing each dye to emit light at different wavelengths.
What is the role of the computer in high-throughput sequencing with capillary electrophoresis?
The computer collects light patterns emitted by the fluorescent dyes and converts them into DNA sequences, handling up to 900 base pairs at a time.
What is next-generation sequencing (NGS) with pyrosequencing?
NGS, such as the Roche 454 system, uses pyrosequencing, where pyrophosphate reactions produce light to sequence DNA fragments attached to beads through emulsion PCR.
How does ATP sulfurylase contribute to pyrosequencing in NGS?
ATP sulfurylase converts pyrophosphate (PPi) into ATP, which is then used in a reaction with luciferase to produce light in a luciferin-luciferase reaction.
What is the role of luciferase in pyrosequencing?
Luciferase converts luciferin into oxyluciferin using ATP, generating visible light that is detected to sequence DNA in pyrosequencing.
What is the principle of NGS (2nd generation sequencing) using the Ion Torrent PGM? Personal Genome Machine
- Utilizes the release of H⁺ ions on a semiconductor
- DNA → Ions → Sequence
- Nucleotides flow sequentially over the ion semiconductor chip
- One sensor per well per sequencing reaction
- Direct detection of natural DNA extension
- Millions of sequencing reactions per chip
What are the key features of 3rd-generation sequencing (3rd GS) using Oxford Nanopore Technologies’ MinION?
- Sensor detects changes in ionic current at nanopore
- nanopore formed in membrane, DNA or RNA pass through
- 10+ kb read lengths
- Error rate ~5%
What is Southern Blotting used for
Used for gene copy number determination, gene mapping, mutation detection, and PCR product confirmation
What are Key Southern Blotting Steps
- Cut DNA with restriction enzymes
- Separate fragments by agarose gel electrophoresis
- Then bascially follow the steps of colony hybridization.
- Using audioradiography or a digital camera, the number of bands will represent gene copy number
What is Northern Blot Analysis used for
To study gene expression by analyzing mRNA
What are the key steps in Northern blot analysis
- Isolate RNA from tissue of interest
- Separate RNA by gel electrophoresis
- Blot RNA onto a nylon filter, UV to fix onto filter.
- Hybridize with a labeled DNA probe
- Detect bands on autoradiograph, showing the presence and size of mRNA
- So it determines whether a gene of interest is expressed ( if binded) as the probe corresponds to it.
How is reverse transcription PCR (RT-PCR) used to study gene expression?
Used to study mRNA levels when detection is below Northern blot sensitivity
