Learning objectives
Explain how advances in recombinant DNA technology have helped scientists study genomes.
1) Protein production -> Protein dissolves blood clots in heart attack therapy, human growth hormone treats stunted growth
2) gene transfer -> for cleaning the ocean contamination by using altered bacteria + gene for pest resistance crop yielding
Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology.
NATURAL FUNCTION OF RESTRICTION ENZYMES
Restriction endonuclease is naturally found in bacteria and protect them from foreign DNA by cutting it up.
Each one is specific in the site that it cuts
HOW THEY ARE USED IN RECOMBINANT TECHNOLOGY
It has an ability to recognise specific sequence of the DNA, By using the same restriction endonuclease in cutting the vector DNA and gene of interest, it allows them to be connect together with ligase
Outline the procedures for cloning a gene in a bacterial cell.
-Gene of interest, Cloning vector, restriction endonuclease, Donor cell
1) cut the vector DNA in the restriction site with restriction endonuclease
2) Extract the gene of interest from the cell with the same restriction endonucelase
3) Insert the gene of interest to the vector gene and connect them with ligase
4) Reintroduce recombinant DNA to the host cell
Describe a cloning vector with an example.
There are 2 cloning vectors are introduced
1) Plasmid ( seperated from bacterial cell's chromosomal DNA) -> pBR 322
-small, circular, self replicating DNA
-one to many copies per cell
- often carriy gene favourable to host
(PBR322)
-4.3kb
-single restriction enzyme sites (useful for insertion)
-ampicilin and tetracycline resistance
-use antibiotic resistance for selection
2) Viral vector -> bacteriophage lambda
- E coli virus
-49kb (circular in host)
-can carry larger fragment
3) Retroviruses -> viruses that infect eukaryotic cells
-Have RNA based genomes
-Carry enzymes that insert viral genome into host cell's genome
-Potentially useful for gene therapy
Describe two techniques to introduce recombinant DNA into eukaryotic cells.
THere are 5 different methods to introduce recombinant DNA into eukaryotic cell
1) Pellet gun (DNA microshot surface)
2) Electroporation (several thousand volts depolarises membrane)
3) microinjection into the nucleus
4) Put cell into lipid preparation to increase permeability of plasma membrane
5) Transfer coupled to vector system
6) CaCl2 + heat allows cell to swell and DNA may enter
Describe the Polymerase Chain Reaction and its application
It is a method to replicate massive amount of gene in a short time.
It has a process of heating->annealing->cooling
First, it has to be heated to denature DNA to seperate each of the strands
Then it should be cooled enough to allow primer to attach to the DNA strand. Primer is composed of complemenatry bases of the DNA sequence. THis process is called annealing
After that a slight increase of temperature allows taq polymerase to replicate the DNA strand. It synthesise in 5'->3' direction
It is used in discovering DNA sequence from the small fragments from the old fossil/ mammoth,
or tiny amount of DNA found in the crime scene.
Describe how DNA technology is used to study the sequence, expression and function of genes
-Knowing the sequence of the gene allows comparison to genes of other species where gene product and function may be known
-To study gene expression, clone gene, make into labelled probe for hybridisation with mRNAs in intact organism. If labelled probe binds, then mRNA transcribed in that tissue, (gene is expressed)
-Microarray : looks for expression of thousands of genes in a tissue sample. Isolate mRNA, copy into DNA by reverse transcription (makes cDNA) using fluorescently labelled nucleotides, apply to microarray (which contains SS DNA for thousands of genes), look for flurescent signal
-In vitro mutagenesis : disable gene (knock-out), reintroduce into cell and observe consequences or use RNA interference to knock down gene expression
Describe how DNA technology can have medical applications in such areas as the development of gene therapy,
human protein production, and the development of pharmaceutical products.
Human protiein production can be used in treating growth stunt or insulin production. Insulin is needed to treat diabetes,
Explain how DNA technology is used in the forensic sciences and to determine paternity.
In genome structure, repetitive DNA exists.Repetitive DNA refers to the different types of short sequences repeated tandem or interspersed in genomeSTR (short tandem repeats) can be 100bp tandems, occurs stretches up to 100kb, most line near centromere or telomere, MINI satellites 6-500bp, MICRO satellites 2-5bp.
-Human DNA fingerprinting uses STR (2-5bp)Each individual has 2 alleles with a certain nuber of repeats, number of repeats varies from individual to individual termed polymorphic (only twins have same number of short repeats)1) DNA is amplified by polymerase chain reaction2) Fragments are separated by gel electrophoresis3) Compare fragment sizes (mother,child, and purpoted father or compare evidence at crime scene with suspect's DNA)
Describe how gene manipulation has practical applications for agricultural work.
CROP IMPROVEMENT
-Transgenic crop development has 3 aims
1) Pest control : insert gene that protects plants from certain pests
2) Weed control : insert gene to protect against herbicide (resistance to herbicide)
3) Crop improvement : insert gene for vitamin or resistance to drought (resistance to drought)
Describe reproductive and therapeutic cloning and why these techniques are controversial.
Reproductive cloning (NUCLEUS TRANSPLANT)
is done by inserting nucleus to nucleus absent egg cell to produce it
It is a production of NEW INDIVIDUAL
Therapeutic cloning (STEM CELL CULTIVATION)
is done by extracting embryo or adult stem cells, cultivate in the lab to produce pluripotent, multipotent stem cell to make into desired cell type, desired cell tissue
NOT PRODUCING ORGANISM JUST PRODUCING CELL/ TISSUE
-> producing controlled individual or cell / tissue is controversial about the border of definition of life
PLUS in the process of extracting nucleus or blastocyte, it is inevitable to destroy the embryo or organism
Discuss the safety and ethical questions related to recombinant DNA studies and the biotechnology industry.
SAFETY -> containment of laboratories, bacterial strains of low viability, limitation of experiments by regulation (national and international), implication
ETHICAL -> concerning extent of human, animal or plant manipulation
It destroys embryos. WHAT CONSTITUTES HUMAN LIFE?
Describe the different types of DNA sequence arrangements.
DNA sequence arrangement
1) Protein coding (1.5%)
2) Repetitive
3) Unclassified (non-coding DNA)
PPT
Gene cloning , PCR and application
DNA Sequencing and In-Situ Hybridisation
Insitu -> is to identify where this gene is expressed
Microarray
Knock out mice
Organism and Therapeutic cloning
Hazards
Genome structure
SPQ
1.
Plasmids and viral vectors are used as cloning vehicles for recombinant DNA work
-Example of plasmid incudes pBR322, which is 4.3kb size, has one restriction enzyme sites, small circular double stranded,
-Example of viral vector is bacteriophages lambda, which is 49kb size with linear genome and can carry larger DNA fragment.
2.
1. Cut the vector DNA's restriction site with restiction endonuclease (restiction ensyme)
2. Cut the gene of interest with the same restriction endonuclease and join together with the DNA ligase
3. Reintroduce the recombinant DNA to the host cells
3.
Plasmids are important in biotechology because they are a vehicle for the insertion of foreign genes into bacteria
4.
You should use the same restriction exonuclease for vector DNA and DNA of gene of interest.
5.
Cloning vector is an agent, such as a plasmid, used to transfer DNA from an in vitro solution into a living cell
-> Cloning vector is a carrier of the gene of interset, which can produce gene or protein in our desired way.
6.
1) Extract plasmid DNA from bacterial cells
2) Cut the plasmid DNA using restriction enzymes
3) Hydrogen-bond the plasmid DNA to non-plasmid DNA fragments
4) Use ligase to seal plasmid DNA to non plasmid DNA
5) Transform bacteria with recombinant DNA molecule
7.
1) Gene transfer -> to plants and clean the toxic environment
2) Protein harvest -> used in therapy
Production of hormones for treating diabetes, and dwarfism/ Production of viral subunits for vaccines/ prenatal testing for carriers of harmful alleles => o
Introduction of genetically engineered genes into human gametes => x
8.
The useful of DNA fingerprinting
A) The sequence is not varied/ the number of STR repeated is varied
B) It is inherited due to genome/ base sequence..?
C)
D) The number of repeats varies widely from person to person or animal to animal
PCR
(Material needed)
-Target gene, Primer (which is complementary to the end of the target gene), nucleotides, taq polymerase
1) Heating : by increasing the temputure, it denatures the DNA H bonding between double strands, seperating the double strand
2) Cooling (Annealing) : by cooling it, primer can form H bonding to the target DNA
3) Rising temperature again to allow taq enzyme (polymerase) can work at optimal temperature
it attaches to the primer and begin polymerase, adding nucleotides to 3' end of primer
Multigene families are collections of identical or very similar genes (a globin, b globins are polypeptides of hemoglobin, and are coded by genes on different human chromosomes)
They are very similar genes but coded from different chromosomes ! (duplicated protein-coding gene)
1) Protein coding gene -> SINGLE or DUPLICATED / code for proetin or protein families / Pseudogenes
2) Repetitive gene -> different types dispersed throughout genome (SINES, LINES, satellite DNA near telomeres and centromeres) ==> used in DNA finger prints
3) Unclassified non-coding gene : unclassified, miRNAs, short interferring RNAs (plants, C, elegans)
Reproductive clonging involves the production of a new organism. It is usually done by nuclear transplatation of a differentiated cell in to an enucleated egg.
--> nuclear transplantation to enucleated egg to make new individual
(Only a small percentage of cloned embryos develop normally to birth)
(Many epigenic changes sounbd be reversed)
Therapeutic cloning involves the production of a range of differentiated cell types from an undifferentiated stem cell
--> Embryonic stem cell (extract stem cells from the inner mass or blastocyst, inevitable to destroy embryonic cell, pluripotent, it can become any type of cell, tissue )
--> Adult stem cell (multipotent, it is difficult to change aSC to iSC, it has limited potential to become a certain cell type)
It can make use of early stage embryonic cell which may be considered a life
When does life begin?