Topic 7 - Modern Genetics Flashcards
Recombinant DNA
DNA that has been formed artificially by combining DNA from different organisms
How can recombinant DNA be produced
1) Isolate required gene
2) Insert gene in a ‘vector’
3) Transformation- gene is delivered into the required cell for protein growth
4) Identify host cells that have taken up the gene
5) grow cells with new genes on a large scale
Gene technology
The manipulation of genes in living organisms
Three ways to isolate the gene of interest
1) Using reverse transcriptase
2) Using restriction endonucleases
3) The Gene Machine
What does detergent do?
- Isolates the DNA from a sample
- Breaks down cell membranes
- Protein may then be removed using digestive enzymes
Reverse transcriptase method of isolation
- RNA taken from a cell that produces required protein
- Reverse transcriptase found in retroviruses like HIV. This catalyses reaction where cDNA is made from mRNA & DNA nucleotides (results in a single strand of cDNA)
- DNA polymerase and free nucleotides used to produce double strand of cDNA
Restriction endonuclease method of isolation
- Gene can be removed from the chromosome using restriction enzymes (restriction endonucleases)
- Different restriction enzymes cut the DNA at a different base sequence (called recognition sequence)
- Restriction enzymes made by bacteria, used to destroy DNA of bacteriophages
Gene machine method
- Examine amino acid sequencing in primary structure of desired protein then work backwards to work out mRNA sequence required to produce this and then the DNA sequence
- Computer can create small section of overlapping DNA called oligonucleotides which are joined to create desored gene
- PCR then used to amplify this copy
Host cells that take up the vector are said to be…
Transformed
How does transformation occur
- Place the host cells in ice cold calcium chloride solution to make cell walls more permeable
- Plasmids then added and the mixture is heat shocked which encourages cells to take up the plasmids
Three types of vectors
1) Gene gun
2) Liposome wrapping
3) Microinjection
What is a gene gun
- Used to produce recombinant DNA by shooting the desired DNA into the cell at high speed on minute gold or tungsten pellets
What is liposome wrapping
- technique involving wrapping the gene in liposomes which fuse with the cell membrane & pass throught to the cytoplasm
What is microinjection
- Technique involving injecting DNA into a cell through a very fine micropipette.
How to check if the cell has been transformed?
1) Marker genes can be inserted into the vectors at the same time as the DNA fragment
2) Host cells are grown on agar and each cell divides replicating the DNA, creating a colony of clones
3) the marker gene can either code for antibiotic resistance or can code for fluorescence by using the GFP gene
4) When under UV light only the transformed cells with fluoresce
How are GM plants made?
- Agrobacterium tumerfaciens (usually) which causes tumours in plants
- It contains a plasmid called Ti plasmid which transfers genetic material into plants
- Plants remain totipotent, GM plant tissue can then be grown through tissue culture
- Tissue culture transferred to a gel with different hormones to trigger development of roots and shoots
- Then clone the plants
Uses of GM crops?
- Improve yield
- Improve disease resistance
- Improve production (balance of fatty acids to prevent oxidation of soya products)
GM soya beans
- made resistant to herbicides
- Fatty acid balance
Whats a knockout mouse?
A laboratory mouse in which one or more genes have been turned off or ‘knocked out’
What is a transgenic animal?
One whose genome has been changed to carry genes from another species or to use techniques for animal genome editing for specific traits.
Genome?
All genetic material in an organism
Polymerase Chain Reaction (PCR)
Technique for the amplification of DNA in vitro (outside the body)
What does PCR require?
1) DNA polymerase (duplicates DNA, Taq found in hot springs thus stable in high temperatures
2) Primers x2 (piece of single stranded DNA complementary to specific target sequence) allows you to amplify any
3) Free nucleotides to make amplified DNA
4) Original DNA strand needed to be replicated
PCR process
1) DNA heated to 95c to break hydrogen bonds between chains
2) Cool to 50-65c allows primers to anneal
3) Heat to 72c for DNA polymerase to attach nucleotides
4) Repeat process multiple times
3 steps of PCR
1) Denaturation
2) Annealing
3) Extension
DNA sequencing process
1) The DNA is chopped into smaller pieces
2) Double strands are separated into single strands
3) PCR is used to amplify (replicate) the DNA
4) Terminator bases are added to the strands
5) The tags allow the bases to be read rapidly
Terminator bases
Modified versions of bases, they have a fluorescent tag attached
It stops anymore bases from being added.
DNA sequencing
What happens after the segments have been sequenced?
The raw data is fed into a computer system that reassembles the genome, by looking at overlap between fragments
How can the sequenced information be used?
1) Predicting amino acid sequences
2) Disease management (mutations that cause disease)
3) Study the predisposition of families to certain diseases
DNA profiling
Differences in our DNA that allows us to identify individuals and make a DNA profile (oftern found in introns of DNA)
What are micro and mini satellites?
Short sequences of DNA which are repeated many times within the introns. (found in same positions on each pair of chromosomes but number of repeats changes between individuals)
Micro- 2-6 bases, repeated 5 to 100 times
Mini - 10-100 base sequence, repeated 50 to several hundred times
How is a DNA profile made?
1) DNA is cut into pieces using enzymes
2) Electrical current separates the DNA pieces in a gel
3) Separated DNA pieces transferred to a membrane
4) Chemicals make the bands visible on the membrane
Gel electrophoresis
- Fragments added to wells of an agarose gel
- Current is passed through the gel, as DNA is negatively charged the DNA moves from negative to positive down the gel
- Smaller fragments travel faster, so will move further down the gel in the same amount of time
- Southern blotting then makes the banding pattern more stable - the DNA is transferred to a nylon/ nitrocellulose membrane to be viewed
What is a transcription factor
Proteins which move in from the cytoplasm into the nucleus and bind to specific DNA sites called promoters. Promoters are found at the start of their target gene.
They can either be activators or repressors (inhibit/decrease the rate)
Transcription factors
Activators and repressors
- Activators - help/increase rate of transcription (help RNA polymerase to bind to the start of the target gene and activate transcriptions
- Repressors - inhibit or decrease the rate by preventing RNA polymerase from binding to the target gene, therefore stopping transcription
Give an example of a transcription factor
Oestrogen
- Affects transcription by binding to oestrogen receptor (transcription factor)
- This then moves to the nucleus where it binds to the promoter region before the target gene
- Then acts as a promoter
Stem cells
- Unspecialised cells that can divide and develop into other types of cells
- All contain same genes but during development not all of them are transcribed and translated therefore genes are expressed or switched off
What happens after a gene is expressed?
- mRNA transcribed and translated
- proteins modify the cell
- cell becomes specialised
Types of stem cells
- Unipotent
- Multipotent
- Pluripotent
- Totipotent
Epigenetic control of stem cells
- Zygote stats totipotent becomes pluripotent as it divides
- This occurs because of transcription factors and epigenetic controls switching on/off certain genes
- Development progresses more genes become silenced (not read) and more become specialised
Induced pluripotent stem cells
- Can be produced from adult somatic cells using protein transcription factors
- Can be used for the treatment of disease and are an option to avoid the use of embryonic stem cells
Therapeutic cloning
Experimental technique to produce large quantities of healthy tissue
1) Remove the nucleus from a patient’s normal body cell
2) Transfer it to a human ovum which has had its nucleus removed, fuse them with an electric shock which further causes development to take place
3) Pre embryo starts to develop and divide, producing embryonic stem cells
4) The genetics perfectly match the patient. The stem cells can be collected and cultured in a suitable environment to differentiate into the tissue needed
5) They can then be transferred back into the patient
Epigenetics
Heritable and reversible midifications to the DNA that do not involve changes to the nucleotide sequence
DNA methylation
Addition of a mthyl group to DNA which prevents transcription and affects histone structure to make more/less DNA accessible to RNA polymerase
RNA splicing
1) Gene is transcribed which results in pre- mRNA
2) All introns (non-coding regions) and some exons (coding regions) are removed
3) The remaining genes are joined together by enzyme complexes called splicesomes. The same exons can be joined in a variety of ways to produce several different versions of mature functional RNA
