Lesson 9: Gene Transfer, Regulation, and Biotechnology Flashcards
Genetic information from two different parents by meiosis
Parasexual
- enhances genetic diversity
- unidirectional from donor to recipient
Conjugation was discovered by
Joshua Lederberg and Edward Tatum
Carries genes enabling the transfer of DNA from an F positive to an F negative cell
A circular piece of DNA that can exist as an extra chromosomal element or may integrate into the chromosomes of the host cell
Episome
Possesses the recombinant DNA in all of its body cells
Transgenic organism
Consist of a combination of DNA from more than one gene or more than one species
Recombinant DNA
What are the steps in PCR?
- denaturation-DNA template is separated by using heat (95°)
- annealing-DNA primers will bind to each DNA strand based on complementary base pairs(55°)
- extension-DNA polymerase will create a new strand of DNA that is complementary to the template strand forming a primer on each strand
Where does heat-stable DNA polymerase come for use in PCR?
Isolated from the thermophilic bacteria (T. aquaticus, and archeae P. furiosus)
*taq polymerase
What is needed for PCR?
- DNA template
- taq polymerase
- primers (oligonucliotides)
- nucleotides
- cofactors
- buffers
Is real time PCR qualitative or quantitative?
Quantitative
- uses two fluorescent dies to collect quantitative data
- same components of PCR
- also requires probe, this allows the researchers to monitor the process of the reaction as it occurs
Real time PCR
In real time PCR, the reporter dye is on the _____ and quencher dye is on the ____ end. Called florescence energy transfer.
5’
3’
Two newer DNA sequencing methods
- next generation sequencing
- third generation sequencing (• Single molecule, real-time (SMRT) • Nanopore sequencing)
DNA that will replicate without integrating into a host chromosome such as plasmid or other types of viruses. Serves as a carrier of the DNA segment to be cloned. Carries from donor to recipient cell
Vector DNA
Enzymes that are used to cut genes of interest from a donor cell. Used to digest DNA
Enzymes that cut DNA creating blank cuts or sticky ends , Results in “restriction fragment”
Recognize and cut at palindromes (racecar)
Restriction endonucleases
Separates DNA based on size, charge, shape
DNA can then be transferred from gel to nitrocellulose membrane and probes maybe used to localize DNA sequences of interest
After DNA is cut from the donor it can be pasted into recipient DNA (vector) using ligase
Agarose gel electrophoresis
Transfer of recDNA to cloning host = “__________”
Transformation
Uses electricity to make small holes temporarily in the membrane or wall of the recipient cell
Electroporation
Chemicals can also create temporary holes
Use in Vitro fertilization or can entire nucleus be integrated
Micro injections
Fatty bubbles that can encapsulate DNA. Brought into the cell by endocytosis or fusion with the plasma membrane
Liposomes
This is more common, when you placed the cells in the DNA in the same tube and increased temperature. Transiently allowing the membrane of the cell to be more soluble, then placed on ice to make membrane more rigid to trap the DNA within
Heat shock
Encodes beta-galactosidase, cancleave substrate to turn colony blue
lacZ
Steps for the production of recombinant DNA
- Purify chromosome DNA
- purify the plasmid (vector)
- insert the gene of interest into the vector
- transformation
- Selection of transform cells
Nitrocellulose membrane, a piece of nucleic acid is used as a probe to detect the DNA and bind
Gel electrophoresis
A way to analyze DNA
Separated DNA fragments are transferred from gel to nitrocellulose membrane.
Probes used to localize DNA sequence of interest
Analysis of DNA by Southern blot
Applications of southern blotting/DNA fingerprinting
Analyze evolutionary links, study of endangered species, tell he was related to whom, identify specific genes, diagnosis of infectious disease, detect pathogen’s in food samples, detect genetically modified foods or organisms, forensic analysis
Used to identify specific RNA molecule, it can determine if the specific gene is transcribed. Example is a nerve cell versus a muscle cell. Also determine if pre-mRNA is alternatively spliced. Example fetal cells versus adult cells
Northern blotting
- used to identify specific proteins
- Specific proteins are detected by antibodies. If a protein made at a particular stage of development
Western blotting
What is a DNA hybridization test?
Probes made of nucleic acid‘s bind complementary regions
-reporter Molecules (•Fluorescent dyes, • enzyme linked)
Used to diagnose cause of disease, identify pathogens and genetic disorders
It has that increase his temperature to denature into single strand and then decreases temperature to produce two strands to anneal, different strands with compatible base pairs also aneal only Homologous DNA will attach
DNA hybridization tests (nitrocellulose)
Test used to detect the physical location on a gene
In-situ hybridization test (FISH) (Florence used)
- Probes apply to intact cells
- observed microscopically for the presence and location of specific genetic marker sequences
- effective way to locate genes on chromosomes “Genetic Mapping”
- ex: Test for leukemia
Analysis of DNA by fluorescent in situ hybridization (FISH)
Molecules of immobilized single-stranded DNA fluorescently labeled molecules that are washed over the micro array
-Used to screen for inherited diseases, diagnose infection, monitor gene expression
Genetic screening by using DNA microarrays
Can somatic cell gene therapy be inherited by the offspring?
No, it cannot help the offspring
This helps with symptoms but does not cure disease
Introduces wild type allele into mutant cells via a viral vector
This type of gene therapy could cure disease and prevent transmission
Ethical problems that could be induce from the therapy most problems are trace to the vectors used
Germ line gene therapy
This is heritable
These often have transient expression because the gene is not integrated into the chromosome therefore the gene is only expressed as long as the viral infection persist
Adenoviral vectors
This type of gene therapy randomly integrate causing mutations in the gene insertion cannot be controlled
Retrovirus gene therapy
