13.8 Genome project and gene technology Flashcards
Genome definition
The complete set of genes in a cell
Proteome definition
The full range of proteins produced by cells
Recombinant DNA definition
A cell that has two or more sources of DNA
How is recombinant DNA achieved?
By isolating fragments of DNA and then inserting them into the DNA of another species
Universal definition
The same triplet coded for the same amino acid
Degenerate definition
More than one codon codes for one amino acid
Non overlapping definition
Each base is only part of one triplet
What are the 5 steps in recombinant DNA technology?
- Isolation of genes
- Insertion
- Transformation
- Identification
- Growth/cloning
What are the 3 ways you can isolate a specific DNA fragment
- Conversion of mRNA to complementary DNA using reverse transcriptase
- Using restriction endonucleases to cut fragments containing the desired gene from the DNA
- Creating the gene in a gene machine
Describe how to isolate a DNA fragment using reverse transcriptase
- Free DNA nucleotides bind to single stranded mRNA template cia complimentary base pairing
- Reverse transcriptase joins DNA nucleotides together to form a single stranded cDNA molecule
- DNA polymerase is required to make cDNA double stranded
What are the advantages of using reverse transcriptase?
- mRNA is much easier to obtain
- Bacterial DNA doesnt contain introns
- Using mRNA isolated from cytoplasm means introns have been removed and exons spliced back together
Explain how to isolate a gene using restriction endonucleases
Restrictionon endonucleases definition
Enzymes that hydrolyse DNA at specific recognition base sequences, usually either side of a desired gene
Describe isolation of DNA fragments using the gene machine
- Desired nucleotide sequence fed into the computer
- Synthesis of short sequences of nucleotides
- Assembly of the gene using PCR
- Gene is inserted into the plasmid
Vector definition
A DNA carrier used to transfer foreign DNA into cells
Describe the isolation of genes into a vector
Isolated target DNA fragments inserted into the vector DNA by cutting open the vector DNA using the same restriction endonucleases
This produces complimentary sticky ends between the DNA fragments ad the cut ends of the vector DNA
- Target DNA fragments anneal to the vector DNA by complimentary base pairing between the sticky ends
- DNA ligase is used to join the DNA fragment and vector at the backbone, forming phosphodiester bonds
This forms recombinant DNA
Outline a method for in vivo cloning
- Cut desired gene from DNA of desired organism
- Use restriction endonucleases
OR - Use mRNA from cell of desired organism
- Use reverse transcriptase to form desired DNA
THEN - make artificial DNA with correct sequence of bases using DNA polymerase
- Cut plasmid open with same restriction endonucleases
- use DNA ligase to join the sticky ends together
- return the plasmid back to the bacterial cell
Why do you have to identify transformed host cells?
- Not all vectors take up the target DNA to become recombinant
- Not all host cells become transformed by taking up the recombinant vectors
Marker gene definition
Allows easy identification of cells that have taken up genetically transformed plasmid
What will cells look like if they haven’t been taken up by any plasmid at all using antibiotics
Will be killed by both types of antibiotic
What will cells look like that have taken up the original plasmid using antibiotics?
Resistant to both types of antibiotics
What will cells look like that have taken up the transformed plasmid when using antibiotics
Resistant to one type of antibiotic but not the second
How do you clone in vivo via bacteria?
Reproduce by binary fission
How do you clone in vitro?
PCR
- polymerase chain reaction
Polymerase chain reaction definition
Used to amplify DNA rapidly and efficiently
What are the three stages of PCR
Seperation
Annealing
Synthesis
Describe the process of PCR
- Heat DNA to 95C to break the weak hydrogen bonds between the strands
- Add primers and nucleotides
- Cool to 50C to allow binding of the nucleotides
- Add DNA Taq polymerase
- Heat to 75C
- Taq polymerase joins nucleotides together
- Repeat the cycle many times
Compare in vitro and in vivo cloning
In vivo compared to in vitro:
- Can be used to produce protein or mRNA from the inserted DNA as well as the target gene whereas in vitro can only be used to copy DNA
- Can be used to clone large NDA frogaments whereas in vitro gets unreliable when copying more than 1000 base pairs
- slower
Need to isolate the DNA fragment from the DNA before it can be inserted into the host cell
- Cant be used to copy partly broken down DNA
What are the benefits of recombinant DNA technology
- Develop medical applications such as insulin to treat diabetes
- Develop agricultural applications to produce crops with a higher yield that are resistant to a disease or extreme weather
- Better understand biological processes
What are the concerns regarding DNA recombinant technology
- Antibiotic resistance may spread
- Inserting new genes into a plant could disrupt the other genes producing genetically modified food sources
- Introducing herbicides could spread to wild species when they interbreed, producing super weeds
- Improving health outcomes by recombinant DNA technology is not always available to everyone socially and economically
Somatic gene therapy definition
DNA transfer to our normal body tissues
Germ line gene therapy definition
DNA transfer to cells that produce eggs or sperm
Limitations of somatic gene therapy
- Not all cells take up new DNA
- Not all cells express the DNA allele
- Only some tissue types are accessible
-Multiple treatments may be needed - Body can produce an immune response to the vector
Gene probe
Short single stranded DNA molecule with a complimentary base sequence to the DNA fragment to be located which is radioactive or labelled by a fluorescent molecule
What are the regions called that are used for DNA fingerprinting
Variable number tandem repeats
Where do you find variable number tandem repeats?
Between genes
Explain the process of DNA fingerprinting
- Extract DNA from the nucleus of the cells
- DNA cut into segments using restriction endonucleases
- Must leave VNTRS intact
- DNA fragments separated using electrophoresis
- Mixture put into wells on gel and electric current passed through
- Immerse gel in alkaline solution
Radioactive marker added to hybridise the target fragments - Identify using autoradiography
