Module 11 Flashcards
Gene Cloning
Technique of isolating and making many copies of a gene
Utilized in DNA sequencing, gene editing, DNA probes, and expression of clones
Insertion of DNA into Vector
Requires cutting (restriction endonucleases) and joining of DNA fragments
Restriction enzymes can digest DNA into fragments with “sticky ends” that can bind to complementary strand, while others have “blunt ends”
Complementary DNA (cDNA)
A DNA strand that does not contain introns and only exons (protein-encoding genes), formed from reverse transcriptase on mRNA
DNA Library
Variety of DNA fragments inserted into vectors produces a collection of recombinant vectors
If starting was chromosomal DNA: genomic library
If starting was cDNA: cDNA library
Polymerase Chain Reaction (PCR)
Copies DNA without vectors or host cells, requiring template DNA, oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), and Taq polymerase
3 Steps of PCR
- Denaturation: DNA strands are separated
- Primer Annealing: Oligonucleotide primers bind to DNA strands
- Primer Extension: Nucleotides are added to primers, extending their lengths
Reverse Transcriptase PCR
Used to detect and quantify the amount of RNA in living cells, beginning with RNA isolated from a sample, mixed with reverse transcriptase and a primer which anneals to 3’ end of RNA
Generates ss cDNA which can be used as a template
Real-Time PCR
Used to quantitate the amount of specific gene or mRNA in a sample, carried out in a thermocycler that can determine fluorescent changes using the TaqMan system
TaqMan System
Detects oligonucleotide with a fluorescent reporter molecule and a quencher molecule at either end. As the replicated length increases the fluorescence gets brighter, eventually reaching the cycle threshold
Cycle Threshold (Ct)
The point at which the accumulation of fluorescence in real-time PCR is significantly greater than the background level
Dideoxy DNA Sequencing
Based on DNA replication, a single tube contains all 4 nucleotides along with 4 fluorescently tagged ddNTPs without 3’ OH groups
Sanger sample is loaded onto single gel lane after polymerization, color fluoresces at each location identifying the specific nucleotide at that spot in the sequence
Gene Editing
Altering the sequence of a gene experimentally
CRISPR-Cas Editing Technology
Creates single guide RNA complex made of tracrRNA and crRNA. sgRNA then guides Cas9 to gene sequence using its spacer region which is complementary to the target gene
Cas9 cut can be repaired either through ____ which deactivates gene, or through ____ which allows for the insertion of donor DNA (causing point mutation)
non homologous end joining (NHEJ), homologous recombination repair (HRR)
Northern Blotting
Used to identify a specific RNA in a mixture to determine if a gene is transcribed within a cell type, at a development stage, and also observe if the pre-mRNA is alternatively spliced
Extract RNA mixture, separate via gel electrophoresis, blot onto nitrocellulose fiber, place in solution containing a probe, complementary DNA appears as a dark band
Western Blotting
Used to identify a specific protein in a mixture to determine if a protein is made within a cell type or at a development stage
Protein mixture separated via SDS-PAGE, denaturing them and coating with negative charges. Protein bands in gel are blotted with nitrocellulose which is placed in solution of primary and secondary (conjugated to alkaline phosphatase) Abs, protein of interest revealed as a dark band
Microorganisms in biotechnology are used for:
medicine, agriculture, biological control, and bioremediation
Insulin from Bacteria
Recombinant bacteria form the A and B chains of insulin separately, with each chain stabilized by a methionine and beta-galactosidase which are cleaved allowing for A and B to join
Biological Control
Use of microorganisms or products to alleviate plant problems (e.g. disease from environmental conditions)
Bioremediation
Use of microorganisms to reduce environmental pollutants by transforming structure of toxic pollutant (biotransformation) resulting in biodegradation
Transgenic Organism
An organism containing genes from other individuals/species
Gene Knock-in Mice
Utilizes CRISPR-Cas technology and homologous recombination repair to insert a gene from another organism into the mouse genome at a noncritical site in an oocyte
Molecular Pharming
Creation of transgenic livestock which are encoded to produce important human proteins in their mammary glands
Dolly the Sheep
- A diploid somatic mammary cell from a donor sheep was combined with an unfertilized egg
- The egg nucleus was removed and the cells were fused with an electrical pulse, initiating the development of a zygote
- The embryo was transferred into a surrogate ewe
- Dolly is born! Genetically identical to the donor sheep
Stem Cells
Supply cells that construct bodies from a fertilized egg
Can replenish damaged cells in adults and vary in their capacity to proliferate and differentiate into one or more specialized cell types
Medical Interest in Stem Cells
They allow us to understand the basic genetic mechanisms that underlie development and allow for the treatment of human disease and injury
