recombinant DNA technology Flashcards
1
Q
What is a gene?
A
- the functional units of chromosomes, determine specific characteristics by coding for specific proteins. Traits are the variations of those characteristics. For example, hair color is a characteristic with traits that are blonde, brown, or black, and many colors in between
- the basic physical and functional units of heredity
2
Q
Define and distinguish the terms ‘genetic engineering’ and ‘recombinant DNA technology’?**
A
- genetic engineering- the alteration of an organism’s genotype using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits. The addition of foreign DNA in the form of recombinant DNA vectors generated by molecular cloning is the most common method of genetic engineering.
- recombinant DNA technology- using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest. This method can be used to combine (or splice) DNA from different species or to create genes with new functions. The resulting copies are often referred to as recombinant DNA.
- All in all, genetic engineering is a broader term used to describe all the techniques that can be used for the alteration and modification of DNA, whereas Recombinant DNA Technology is one of the techniques of genetic engineering that is used to produce recombinant DNA.
3
Q
How are genes sequenced?**
A
- gel electropherisis
- create a DNA library (clone fragmens into a plasmid vector)
- sequence each clone
- align overlapping sequences and assemble the genome
- can compare individuals to eachother
4
Q
What is biotechnology?
A
- use of biological agents for technological
advancement - utilizes biological systems, living organisms or parts of this to develop or create different products.
- ex. the development of insulin, crispr
5
Q
Explain molecular and reproductive cloning
A
- molecular cloning: technology fundamental to molecular biology and biotechnology that uses living cells (ex. bacteria, yeast) to make many copies of a DNA sequence of ineterst. plasmids are essential. produces recombinant proteins. create multiple copies of a specific DNA fragment, such as a gene or a regulatory sequence, and to transfer it into a host organism. can be used in gene therapy, gene anaylsis, biomedical applications
- reproductive cloning: the deliberate production of genetically identical individuals.ex. dolly the sheep. creating an organize that is genetically identical to donor organism
6
Q
Describe biotechnology uses in medicine and agriculture
A
- crispr.ex. allergies, alter genome
- gene therapy: can be used to provide genes to individuals who dont have a functional one. ex. build plasmids containing a normal version of the gene that’s nonfunctional in cystic fibrosis
- biomedical applications: vaccines. insulin
- gene anaylsis: build dna to help understand how normal genes work
- gmo crops to be resistant to pesticides and grow bigger
7
Q
Describe the key concepts and principles of molecular cloning
A
- fundamental to molecular biology and biotechnolgy
- uses living cells to make many copies of a DNA sequence of interst
workflow: 1. select plasmid –> 2. target DNA isolation –> 3. create recombinant DNA –> 4. propogate recombinant DNA in bacteria (or another sutiable host) –> 5. screen and select bateria that express your recombinant DNA –> 6. isolate recombinant DNA for further verification and experimentation
8
Q
cDNA synthesis
A
- copy dna
- synthetic DNA that has been transcribed from a specific mRNA through a reaction using the enzyme reverse transcriptase. mrna is isolated and turned back into DNA
- useful for cloning and expressing specific genes
- create cDNA libraries, enabling them to study sequences specific to a given tissue and develop shareable databases
- contains only the expressed genes of an organism, excluding introns (non-coding regions) and regulatory sequences. This makes cDNA particularly useful in genetic engineering, molecular biology, and gene expression studies.
9
Q
What is PCR?
A
- polymerase chain reaction
- DNA amplification in a test tube
- a technique used to amplify small segments of DNA (specific sequences)
- makes billions of copies of a specific DNA fragment or gene, which allows detection and identification of gene sequences using visual techniques based on size and charge.
10
Q
what is restriction digest
A
- technique used in molecular biology to cut DNA into smaller fragments using specific enzymes known as restriction enzymes
- Restriction enzymes, also called restriction endonucleases, recognize specific, short nucleotide sequences in the DNA and cleave the DNA at or near these sites.recognize unquie sequences
- combining a DNA sample with one or more restriction enzymes in a reaction buffer that provides the necessary conditions for the enzyme activity. then incubate. afetr digestion, dna fragments can be separted using gel electropherisis and analyzed
11
Q
what is bacterial transformation.
A
- bacteria take up foreign DNA from their surrounding environment. This genetic material is often in the form of plasmids,
-Once inside the bacterial cell, the foreign DNA can be incorporated into the host genome or can remain as an independent plasmid - f the foreign DNA confers a new trait, such as antibiotic resistance or the ability to metabolize a new substrate, the recipient bacterium can express this trait and pass it on to its descendants.
12
Q
Define genome editing
A
- a menthod used to alter an organism’s DNA by leveraging the cells DNA repair mechanisms
- during cell division, if there is an error, these repair mechanisms will stop division and fix. they will correct mutattions,insert dNA sequences (ex. GFP tagging), and remove DNA sequences (ex.create knock outs = loss of protein function)
- uses recombinant enzymes (endonucleases) to cleave and alter DNA sequences. tools include: zinc finger nucleases, transcription activator-like effoector nucleases (TALENs), and CRISPR
13
Q
Compare and contrast the mechanisms by which different genome editing tools edit DNA
A
ZNFs and TALENs use FOK1. CRISPR uses bacterial immunity
14
Q
Explain how CRISPR is used naturally by cells and in the context of biotechnology and healthcare.
A
- Bacteria capture and store short, unique fragments of viral DNA in their CRISPR arrays during past infections.
- If the same virus attacks again, the bacteria use these stored sequences to generate RNA molecules (CRISPR RNAs or crRNAs) that guide Cas proteins to the specific location on the viral DNA.
- Cas proteins then act like molecular scissors, cutting the viral DNA and preventing the infection.
15
Q
what is knockout
A
a gene that is no longer functional. knocks out the function
