Chapter 7 Flashcards
What are restriction enzymes? Why are they important biologically and in the lab?
Tiny molecular scissors that can cut DNA. They work by recognizing specific sequences of letters in the DNA code and cutting the DNA at those specific points.
They are important biologically as bacteria defense mechanisms against viruses (bacteriophages) and in the lab for genetic engineering and studying genes.
What advantage do “sticky end” restriction enzymes have over “blunt end” restriction enzymes for cloning?
“Sticky ends” promote a more stable and specific joining of DNA fragments, making them easier for them to stick together during the ligation process in cloning.
What are the components of a plasmid? Why is each part necessary?
Origin of Replication (ori):
- allows the plasmid to replicate independently of the host cell’s chromosomal DNA
Selectable Marker:
- provides phenotypic trait (usually antibiotic resistance)
Multiple Cloning Site:
- contains multiple unique restriction enzyme recognition sites for researchers to insert genes
Reporter Gene:
- ex: GFP (green fluorescence)
Terminator Sequence:
- helps terminate transcription of the inserted genes
Promoter Region:
- may contain a promoter region that initiates the transcription of the genes in the plasmid
How do you select for bacteria that took up your plasmid with your gene of interest (GOI)?
By incorporating a selectable marker in the plasmid
What is a competent cell? How do you make competent cells?
A bacterial cell that has been treated to increase its ability to take up foreign DNA, such as a plasmid containing a gene of interest.
Making competent cells involves creating an environment that enhances the permeability of the bacterial cell membrane, allowing it to more efficiently absorb foreign DNA during a process called transformation.
What are some applications of DNA cloning?
Recombinant Protein Production
Gene Therapy
Biomedical Research
Production of Transgenic Organisms
DNA Sequencing and Genome Mapping
Vaccine Development
Forensic Analysis
What is cDNA? How is it made? Why is it useful?
cDNA (“copy” or complementary DNA) is a synthesized DNA molecule that is complementary to a specific RNA template. Made by DNA Isolation, Priming, Reverse Transcription, cDNA Purification and Amplification (optional)
Huge for DNA cloning and studying gene expression
What is PCR? Why is it useful?
Polymerase Chain Reaction is a powerful molecular biology technique used to amplify and replicate specific DNA sequences.
DNA AMPLIFICATION is huge for all of the above mentioned applications for DNA Cloning ^
What is the purpose of primers in PCR?
Primers serve as short, single-stranded DNA sequences that are crucial for the initiation of DNA synthesis. They play a fundamental role in guiding DNA polymerase to the specific region of the DNA template that needs to be amplified.
What are expression vectors? How are restriction sites used in vectors? What are operators and repressors and how are they used in vectors? What are inducers and how are they used in vectors? What is the application of expression vectors?
Expression vectors are specialized DNA molecules used in molecular biology to introduce, express, and produce a specific gene or protein in a host organism, typically a bacterium, yeast, or mammalian cell.
What are operators and repressors and how are they used in vectors?
Operators: the operator is a DNA sequence located near the promoter region of a gene.
- Acts as a binding site for repressors to turn genes on or off
Repressors: a repressor is a regulatory protein that can bind to the operator region of a gene.
- Inhibit gene expression by preventing RNA Polymerase from binding
What are inducers and how are they used in vectors?
Molecules that can activate gene expression by modulating the activity of regulatory proteins, such as repressors, in a gene expression system. In the context of vectors, such as plasmid vectors used in molecular cloning and gene expression studies, inducers are often employed to control when and to what extent a target gene is expressed.
What is the application of expression vectors?
Vectors are designed to carry and express a specific gene of interest in a target host
What is GFP and what is it used for? How are variants of GFP made?
Green Fluorescent Protein. Used for cellular and molecular imaging, gene expression studies, and biomedical research. Made by introducing specific mutations or modifications to the GFP gene, which is then expressed in living cells.
Be able to outline the steps involved in Sanger sequencing. What are the applications? What is the purpose of the four different fluorescent tags?
DNA Denaturation
Primer Annealing
DNA Synthesis
Termination & Separation
Electrophoresis
Detection
Data Analysis
Used for genome sequencing, forensic DNA analysis, pharmaceutical research and much more!
4 Different Tags correspond to 4 different bases
Be able to outline the steps involved in Western Blotting. What are the applications?
Protein Extraction
Protein Quantification
SDS-PAGE (Polyacrylamide Gel Electrophoresis)
Protein Transfer
What are the applications of CRISPR/CAS9? What are some advantages of using CRISPR/CAS9?
Super Advanced Gene-Editing Technology! Much more precise, versatile, and cost effective
Why is DNA cloning important? (think PCR)
Individual DNA fragments can be isolated and cloned for detailed analysis and for expression of proteins in various applications
What does the Type II Restriction Endonuclease enzyme do?
Cleaves DNA at specific base sequences
- Some make blunt end cuts, others create sticky ends
What does the DNA ligase enzyme do?
Joins two DNA molecules or fragments
What does the DNA Polymerase enzyme do?
Fills single-stranded gaps in duplex DNA by stepwise addition of nucleotides to 3’ ends
What does the Reverse Transcriptase enzyme do?
Makes a DNA copy of an RNA molecule (RETROVIRUSES)
What does the Polynucleotide Kinase enzyme do?
Adds a phosphate to the 5’ OH end of DNA
What is Sequence Assembly?
“Shredding” apart a bunch of DNA so that we can assemble tiny sequences
What are “Reads”?
Groups of sequences you get off the sequencing machine (one line of DNA of many)
What is a contig? How is it different from a consensus sequence?
A contig is a continuous stretch of DNA that has been assembled from overlapping DNA fragments.
A consensus sequence can be generated from multiple contigs that overlap or represent alternative versions of a specific genomic region. This process involves identifying the most common base at each position, resolving discrepancies, and producing a single, unified sequence that represents the best estimate of the true genomic sequence
- The goal is to have one of our contigs become our consensus sequence!
What is read coverage? Why is it important?
Coverage is the number of repeats in bases when the individual sequences are matched up. Higher coverage = more confidence in results.
Why are repetitive sequences difficult to sequence?
Because there may be multiple ways to assemble sequences (too many contigs)
What is a microarray? How might microarrays be useful for learning about (for example) the effectiveness of a drug?
A tool that can detect the expression of thousands of genes simultaneously.
mRNA ->
cDNA & Tagging ->
Hybridize to chip ->
rinse, read
Microarray datasets can potentially determine the appropriate drug dose that can maximize its therapeutic effect. In clinical trials, microarrays can be used for early detection of any toxicity or any side-effects of a drug or a drug dose in order to provide rapid, sensitive, and safe treatments.
What is precision medicine? What benefit does it offer over traditional medicine? What are some possible complications of precision medicine?
Precision medicine is a way health care providers can offer and plan specific care for their patients, based on the particular genes, proteins, and other substances in a person’s body.
Ethical Concerns
Data Security & Privacy
Side Effects
Cost & Accessibility
