Flashcards in Biotechnology Deck (41):
What is biotechnology?
Any technological application that uses biological systems/ living organisms
Uses for biotechnology?
Healing: vaccines, medications, diagnosis of disease, gene therapy
-DNA and protein analysis to identify relationships and predict how species evolved ( evolutionary theory)
- epigenetics, investigating relationship between environment and gene expression
What are the 11 biotechnological techniques and tools?
Polymerase chain reaction
Genetic engineering (recombinant DNA)
Define gene probing?
A single stranded DNA or DNA used in genetic engineering to search for a particular gene or other DNA sequence
Define DNA profiling?
a technique that uses the banding patterns of DNA fragments as means of identification; a DNA fingerprint is unique to a particular individual; also called DNA fingerprinting
Define DNA sequencing?
The determination of the precise order of nucleotides (nucleotide sequence) in a sample of DNA.
Define polymerase chain reaction ?
(PCR) A technique used in molecular biology for producing multiple copies of DNA from a sample; used in DNA fingerprinting and in identifying diseases
Process of producing clones of organisms or clones of organisms or copies of cells or DNA fragments (molecular cloning)
Define genetic engineering?
(Recombinant DNA) , the procedures used to produce recombinant DNA; involve introducing DNA into a cell from a different type of organism or DNA that has been modified in someway
Define restriction enzymes ?
An enzyme that cuts strands of DNA at a specific sequence of nucleotides
Define ligase enzymes?
An enzyme that can catalyse process of joining short strands of DNA during replication ( ligation?)
In bacterial cell, small circular strands of DNA distinct from the main bacterial genome, composed of only a few genes and able to replicate independently within cells
Define transgenic organisms?
An organism that has had DNA from another species introduced into it artificially.
Where do restriction enzymes come from and what is their role there?
They come from in bacteria where they restrict the duplication of infecting viruses by cutting up the viral DNA
How are restriction enzymes used by scientists?
Scientists use them to cut DNA at recognition sites and restrict the duplication of bacteriophages
What are the 2 types of ends that restriction enzymes can make?
Blunt ends and sticky ends
Describe blunt ends? ( made by restriction enzymes)
Straight cuts made when a restriction enzymes make a clean break across the two strands of DNA. Both strands terminate in a base pair, it is harder to recombinate with them.
Describe sticky ends? ( made by restriction enzymes)
Made when restriction enzymes produce a staggers cut resulting in fragments with sticky ends, sticky ends is a stretch of unpaired nucleotides in the DNA molecule that overhang at the break in the strand
What is a ligase enzyme and how is it useful to biotechnology?
a ligase enzyme is an enzyme able to join of recombine, seperate pieces of DNA. It’s found in bacterium.
It’s useful in bio tech because it is only in the presence of ligase that single stranded breaks in phage DNA could be repeated= enables scientists to attempt own recombination experiments recombining DNA from different organisms, including different species
Purpose of identifying genes?
To be able to extract genes for further analysis and processing.
Ie. comparison of code to establish evolutionary relationships and diagnosis of genetic disease-> treatment plans
Describe genetic probing?
- sample of DNA to be analysed is mixed with radioactive nucleotides and free nucleotides
- DNA replication is initiated (in lab conditions)
Describe DNA profiling?
- separation of genes within a genome based in base pair length
- restriction enzymes are used to cut DNA into individual genes
- use gel electrophoresis to form a DNA fingerprint
Define gel electrophoresis?
The process of using an electric current to seperate DNA across an agarose gel.
- extract DNA from cells
- cut DNA to small pieces w/ restriction enzymes
- DNA is inserted at wells on negative end
- add electric current and strands move towards positive end. Small fragments move further
- agarose gel is porous = DNA moves through spaces
Outline the process of DNA sequencing?
( the Sanger method)
1. Denaturing- DNA sample is collected; specific gene is extracted using restriction enzyme. Heat is used to denature the DNA strand. Template strand is exposed. Copies are made by polymerase chain reaction (PCR)
2. Annealing- primer (known segment of DNA) is added. This indicates the site at which DNA polymerase is to attach and begin copying the DNA.
3. Elongation- DNA polymerase copies the template strand, adding free nucleotides (complementary to the template)
4. Termination- when ddNTP (A,T,C,G) is added, the copying process ends and the new strand of DNA is released .
- DNA segments of varying lengths can be then seperate using gel electrophoresis. Each DNA segment is a different length, by reading nucleotide order a sequence can be identified.
Uses of DNA sequencing?
- identify location of a specific mutation
- genetic counselling
- establishes evolutionary trends/ relationships
What is a recognition site?
A specific sequence of nucleotides at which an enzyme cuts a strand of DNA
What is a plasmid?
- circular, double stranded DNA found in bacteria capable of independently replicating apart form chromosomal DNA.
How are plasmids used to make proteins
- plasmid is selected based in whether or not DNA can be easily inserted into it.
-cut using the same restriction enzyme used to extract desired gene (ie human insulin)
-DNA segment is inserted into plasmid- DNA ligase bonds to form = recombinant DNA
- recombinant DNA is inserted into a transgenic organism
- transgenic organism capable of transcribing the recombinant DNA and thus performs protein synthesis to manufacture the desired gene
What are the 3’ prime and 5’ prime ends ends of the DNA strands important?
3 and 5 prime indicate the carbon number in the DNA sugar backbone, it gives DNA strand a direction.
Formula for finding how many strands after a certain number of cycles?
2^(no. Of cycles)
Two to the power of (Number of cycles)
Why are primers added to the DNA strand before DNA polymerase extends the strand?
It acts binding site for DNA polymerase so DNA replication can happen.
What advantages does PCR have over gene cloning?
PCR clones just DNA. Cloning is whole cell. PCR is more efficient and less costly
How can recombinant insulin be made ( brief )
Gene for human insulin is isolated from pancreatic cells. Human gene is inserted into vectors such as plasmid which is then inserted into bacteria or yeast. Insulin gene is then cloned in the bacteria or yeast. Resulting insulin percusor formed from the gene is extracted from the bacteria or yeast and purified for pharmaceutical use by diabetes patients.
What are the steps of the polymerase chain reaction process?
Denaturing- hydrogen bonds of the two strands of the target sequence seperate after being heated.
Annealing- short primer attach/ anneal to each of the two DNA strands at the 3’ ends to act as starting points for elongation
Elongation- sample is heated and starting at primer DNA polymerase copies and extends the strands adding complementary nucleotides to the template
Cycle ends and two DNA strands are formed, identical to the original DNA target sequence. And this process continues
Stages of cloning?
1. Grow human growth cell in culture Eg. Nerve cell for myelin sheath or pancreatic cells for the insulin gene. Human cells contain the gene of interest gene X
2. Isolate DNA from cultures human cell. Cut gene x from rest of DNA, using a restriction enzyme to create sticky ends
3. Isolate suitable plasmid vector from bacterial cell
4. Cut plasmid with same restriction enzyme used to cut gene x. Producing a plasmid with matching sticky end to the gene
5. Gene x is inserted into the plasmid by mixing the gene and the plasmids together. Some plasmids take up gene x. The sticky ends of the plasmid base pair with the sticky ends of gene x.
6. DNA ligase joins together, the sticky ends of the plasmid and the gene by covalent bonds
7. Recombinant plasmid is put back in bacteria. Plasmid added to growing bacterial culture. Some bacteria will take up the plasmid by the process of transformation.
8. Recombinant bacteria grown on an agar plate. Bacteria multiply and reproduce many copies of gene x. Gene has been cloned. Colonies of bacteria containing blonde gene can be identified and gene can be isolated for scientific or medical use.
What is the human genome project?
International research Effort aimed at mapping the location of the genes in all 46 chromosomes in the human genome
Complete set of genetic information of an organism
What is a phage
Bacteriophage; virus that infects bacteria
What is a vector
Bacterial plasmid, viral phage or other such agent used to transfer generic material from one cell to another
Two diseases gene therapy is used for ?