Genetic Technology Flashcards
What is the aim of genetic engineering?
To remove a gene (or genes) from one organisms and transfer it to another so that the gene is expressed in its new host
What is recombinant DNA?
- It is DNA made by joining pieces from two or more different sources
- The DNA that has been altered by this process and which now contains length of nucleotides from two different organisms is called recombinant DNA (rDNA)
- The organism which now expresses the new gene or genes is known as a transgenic organism or a genetically modified organism (GMO)
Why is genetic engineering important?
- It provides a way of overcoming barriers to gene transfer between species
- Unlike selective breeding where whole sets of genes are involved, genetic engineering often results in the transfer of a single gene
What is the overview of gene transfer?
- GMO produced
1. The gene that is required is identified and it may be cut from a chromosome, made from mRNA by reverse transcription or synthesised from nucleotides
2. Multiple copies of the gene are made using PCR
3. The gene is inserted into a vector, which delivers the gene to the cells of the organisms e.g. plasmids, viruses and liposomes
4. The vector takes the gene into the cells
5. The cells that have the new gene are identified and cloned
What is needed for gene transfer?
- Enzymes, such as restriction endonucleases, ligase and reverse transcriptase
- Vectors, including plasmids and viruses
- Genes coding for easily identifiable substances that can be used as markers
What are restriction endonucleases?
- A class of enzymes from bacteria which recognise and break down the DNA of invading viruses known as bacteriophages (phages for short)
- Bacteria make enzymes that cut the phage DNA into smaller pieces
What is the function of restriction enzymes?
- These enzymes cut the sugar-phosphate backbone of DNA at specific places within the molecule
- This is why they are known as endonculeases (‘endo’ means within)
What is the role of restriction enzymes in bacteria?
To restrict a viral infection
How does each restriction enzyme work?
- Each restriction enzyme binds to a specific target site on DNA and cuts at that site
- These target sites, or restitution sites, are specific sequences of bases
- E.G. The restriction enzyme, BamHI always cut DNA where there is a GGAATCC sequence on one strand and its complementary sequence CCTAGG on the other
How is DNA protected from such an attack by restriction enzymes?
- By Chemical markers
- Or by not having the target sites
How do restriction enzymes cut?
- Straight across the sugar phosphate backbone to give blunt needs
- In staggered fashion to give sticky ends
What are sticky ends?
- Short lengths of unpaired bases
- They are known as sticky ends because they can easily form hydrogen bonds with complementary sequences of bases on other pieces of DNA cut with the same restriction enzyme
- When long pieces of DNA are cut with a restriction enzyme, there will be a mixture of different lengths
How do you find the specific piece of DNA required?
- Separate the lengths of DNA using gel electrophoresis and using gene probes
- Multiple copies of the required piece of DNA can be made using PCR
Why is gene tech important?
- Now that many proteins have been sequenced, it is possible to use the genetic code to synthesise DNA artificially from nucleotides rather than cutting it out of chromosomal DNA or making it by reverse transcription
- Genes, and even complete genomes, can be made directly from DNA nucleotides without the need for template DNA
- Scientist can do this by choosing codons for the amino acid sequence that they need
- The sequence of nucleotides is held in a computer that directs the synthesis fo short fragments of DNA
- These fragments are then joined together to make a longer sequence of nucleotides that can be inserted into plasmids for use in genetic engineering
- This method is used to generate novel genes that are used in e.g. the synthesis of vaccines
How do you get a new gene into a recipient cell?
A vector has to be used
What are plasmids?
- A vector used
- Small, circular pieces of double stranded DNA
- Plasmids occur naturally in bacteria and often contain evens for antibiotic resistance
- They can be exchanged between bacteria. even between different species of bacteria
- If a genetic engineer inserts a piece of DNA into a plasmid, then the plasmid can be used to take the DNA into a bacterial cell
How to you obtain the plasmids from bacteria?
- Bacteria containing them are treated with enzymes to break down their cell walls
- The ‘naked’ bacteria are centrifuged, so that the relatively large bacterial chromosomes are separated from the much smaller plasmids
How is the circular DNA of the plasmid cut open? What is this also used for?
- A restriction enzyme
- The same enzyme as the one side to cut out the gene should be used so that the sticky ends are complementary
What happens if a restriction enzyme is used that gives blunt ends?
-Sticky ends need to be attached to both the gene and plasmid DNA
What happens to the open plasmids and length of DNA?
- They are mixed together
- Some of the plasmid sticky ends pair up with the sticky ends of the new gene (hydrogen bonding)
What is DNA ligase used for?
- To link together the sugar phosphate backbones of the DNA molecule and the plasmid, producing a closed circle of double stranded DNA, containing the new gene
- This is now recombinant DNA
How can bacterial plasmids be modified to produce good vectors or made artificially?
- The pUC group of plasmids have:
1. A low molecular mass, so they are readily taken up by bacteria
2. An origin of replication so they can be copied
3. Several single target sites for different restriction enzymes in a short length of DNA called a polylinker
4. One or more marker genes, allowing identification of cells that have taken up the plasmid
What other vectors are there to plasmids?
- Viruses
- Liposomes, which are tiny spheres of lipid containing the DNA
How do you get the plasmids into the bacteria / get the bacteria to take up the plasmids?
- The bacteria are placed in a solution with a high concentration fo calcium ions
- Then cooled and given heat shock treatment to increase the chances of the plasmids passing through the cell surface membrane
- A small proportion of the bacteria, perhaps 1% take up plasmids with the gene, and are said to be transformed
- The remaining either take up plasmids that have closed without incorporating a gene or do not take up any plasmids at all
How do you identify the bacteria with recombinant DNA?
- Spread bacteria on agar plates each containing an antibiotic
- So if, for example, the insulin gene has been inserted into the plasmid at a point in the gene for tetracycline resistance in pBR322, then any bacteria which had taken up plasmid with the recombinant DNA would not be able to grown on agar contains tetracycline
Why is this important to identify the bacteria with recombinant DNA?
Important to identify which bacteria have been successfully transformed so that they can be used to make the gene product
What happens after the plasmids have been taken up?
- DNA polymerase in bacteria copies the plasmids
- The bacteria then divide by binary fission so that each daughter cell has several copies of the plasmid
- The bacteria transcribe the new gene and may translate it to give the required gene product e.g. insulin
What is a form of diabetes caused by?
- One form of diabetes mellitus is caused by the inability of the pancreas to produce insulin
- Before insulin from GM bacteria became available, people with his from of diabetes were treated with insulin started from the pancreases of pigs or cattle
- Then came the idea of inserting the gene for human insulin
What were the problems to producing insulin?
- Locating and isolating the gene coding for human insulin from all the rest of the DNA in a human cell
- Instead of cutting out the gene from the DNA in the relevant chromosome, researchers extracted mRNA
What is the initial step in insulin production?
- Extract mRNA for insulin the pancreatic beta cells, which are the only cells that express the insulin gene
- These cells contain large quantities of mRNA for insulin as they are its only source in the body
Why do they used insulin mRNA over insulin DNA?
- There are several copies of the mRNA without the non-coding sort of the DNA available which have already been transcribed
- DNA is a long molecule and hard to find wanted section
What happens to the extracted mRNA? What is reverse transcriptase and DNA polymerase?
- The mRNA is then insulated with the enzyme reverse transcriptase (this comes from a group fo viruses called retroviruses)
- The enzyme reverses transcription using mRNA as a template to make a single stranded DNA (cDNA)
- These single stranded DNA molecules using DNA polymerase to assemble nucleotides to make the complementary strand
- The genetic engineers now had insulin genes that they could insert into plasmids to transform the bacterium
What is the advantage of using GM insulin?
- There is a reliable supply available to meet the increasing demand
- Supplies are not dependent on factors such as availability through the meat trade
- Can also change nucleotide sequence of the insulin gene to give molecules with different aa sequences to give e.g. act faster or slower
What are is GFP as a genetic marker?
- GFP (green fluorescent protein) that uses enzymes that produce fluorescent substances
- Enzymes obtained from jelly fish make protein GFP, that fluoresces bright green in UV light
What is the concern of using antibiotic resistance genes as markers?
-Antibiotic genes may spread to other bacteria, potentially producing strains of pathogenic bacteria leading to diseases that may be untreatable
How does GFP genetic markers work?
- The gene for the enzyme is inserted into he plasmids
- To identify bacteria that have taken up the plasmid, need to shine UV light onto them
- Glow green = GM ones and the same marker gene can be used in a range of organisms
What is Beta-glucurondiase (GUS) work as a genetic marker?
- Originates from E.Coli
- Any transformed cell that contains this enzyme, when incubated with seem specific colourless or non-flurescnet substrates, can transform them into coloured or florescent product
- Especially useful in detecting the activity of inserted genes in plants
What does a promoter do?
- Controls expression of a gene
- The region of DNA to which RNA polymerase bind and starts transcription
Why are promoters important in genetic technology?
- If you want the gene that we are going to insert into a bacterium to be expressed, then you also need to insert the appropriate promoter
- When bacteria were first transformed to produce insulin, the insulin gene was inserted next to the beta galactosidase gene so they shared a promoter
- The promoter switched on the gene when the bacterium needed to metabolise lactose
- So if the bacteria were gown in the medium containing lactose but no glucose, they synthesised both beta galactodisae and human insulin
What are the functions of promoter?
- The promoter allows RNA polymerase to bind to DNA and it also ensure that it recognises which of the two DNA strands is the template tsrand
- Within the sequence of nucleotide in the promoter region is the transcription start point (the first nucleotide of the gene to transcribed)
- In this way the promoter can be said to control the expression of a gene and can ensure a high level of gene expression
- In Eukaryotas, various proteins known as transcription factors are also required to bind to the promoter region or to RNA polymerase before transcription can begin
What is gel electrophoresis?
- A technique that is used to separate different molecules
- Used in analysis of proteins and DNA
What is involved in gel electrophoresis? What does movement depend on?
- Place a mixture of molecules into wells cut into agarose gel and applying an electric field
- The movement of charged molecules within the gel in response to the electric field depends on net (overall) charge, size, composition of the gel
How does net overall charge affect ge?
- Negatively charged molecules move towards the anode
- Positively charged molecules move towards the cathode
- Highly charged molecules move faster than those with less overall charge
How does size affect ge?
-Smaller molecules move through the gel faster than larger molecules
How does composition of gel affect ge?
- Common gels are polyacrylamide for proteins and agarose for DNA
- The size of the ‘pores’ within the gel determines the speed with which proteins and fragments of DNA move
How does electrophoresis of proteins occur?
- The charge on proteins is dependent on the ionisation of the R groups on the amino acid residues
- Some R groups are positively charges (-NH3+), (-COO-)
- Whether these R groups are charged or not depends on the pH
- When proteins are separated by electrophoresis, the procedure is carried out at a constant pH using a buffer solution
- Usually proteins have a net negative charge
How is ge used in polypeptides?
- Ge has been used to separate the polypeptides produced by different alleles of many genes
- E.G Allozymes are variant forms of enzymes produced by different alleles of the same gene
How can ge be used to diagnose sickle cell anaemia?
- There are many variant of haemoglobin
1. Adult haemoglobin is composed of four polypeptides (2 alpha-globins and 2 beta globins)
2. In sickle cell anaemia a variant of beta globin has an amino acid with a non-polar R group instead of one with an R group that is charged
3. These two variants can be separated by electrophoresis because they have different net charges
4. This means that haemoglobin molecules in people who have sickle cell anaemia have a slightly lower negative charge than normal haemoglobin
5. Therefore the molecules do not move as far through the gel as molecules of normal haemoglobin - The test to find out whether someone carries the sickle cell allele makes use of this difference
What is electrophoresis of DNA?
- DNA fragments carry a small charge thanks tot he negatively charged phosphate groups
- In DNA electrophoresis, these fragments move through the gel towards the anode
- The smaller the fragments, the faster they move
How is electrophoresis of DNA used?
Genetic profiling (fingerprinting) in forensic science
What are VNTRs?
- A region of DNA that is known to vary between different people
- These regions often contain variable numbers of repeated DNA sequences are are known as variable number tandem repeats
- Only identical twins share all their VNTR sequences
