Gene Technology Flashcards
What has automated sequencing led to?
automated sequencing of genomes has led to discovery of many new species.
Some species may look very similar but analysis of their genome shows differences in the DNA bases and therefore differences in the mRNA bases and amino acid sequences.
Genome to Proteome
If researchers know which genes are present, they can determine which proteins can be produced via gene expression.
It is therefore possible to determine the full range of proteins produced by cells (PROTEOME)
Applications of genome sequencing
identification of potential antigens for use in vaccine production.
What has knowledge of the genome led to?
increased study of non-coding DNA and regulatory DNA (e.g. transcription factors, siRNAs, miRNAs, tRNAs)
These make up most of the DNA.
For example, in humans, it is now estimated that 80% of this non-coding DNA is involved in regulating the expression of protein coding genes which only make 1% of our entire genome.
What is recombinant DNA?
a cell having two or more sources of DNA.
This can be achieved by ISOLATING fragments of DNA and then INSERTING this DNA into another organism
(recombinant is also referred to as transgenic or a genetically modified organism (GMO))
Why is recombinant DNA technology possible?
the genetic code is:
universal
non overlapping
degenerate
transcription and translation are also universal
What are the 5 steps in recombinant DNA technology?
- Isolation of genes
- Insertion
- Transformation (transfer into microorganisms)
- Identification
- Growth / cloning
Isolation using reverse transcriptase
makes cDNA from mRNA
Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing.
Reverse transcriptase joins DNA nucleotides together to form a single stranded cDNA molecule.
DNA polymerase is required to make cDNA double stranded
Advantages of using reverse transcriptase
- mRNA is much easier to obtain
- introns have been removed (bacteria can’t splice)
- produce protein in large amount
Isolation using restriction endonuclease
Different restriction endonucleases hydrolyse DNA at different specific recognition sequences because the shape of the sequence is complementary to the enzymes active site.
These recognition sequences are often palindromic whereby the base pair read is the same in opposite directions (DNA is antiparallel).
The DNA sample is incubated with the specific restriction endonuclease(s), which hydrolyses the DNA into fragments wherever the recognition sequence appears.
If the target gene has recognition sequences before and after the target gene, the fragments will contain the desired gene
What are Restriction endonuclease ?
enzymes that hydrolyse DNA at specific recognition (base) sequences.
These are usually either side of a desired gene
What happens if the recognition sequence for the selected restriction endonuclease occurs within the DNA fragment you want to isolate?
will cut the gene and it will not code for a functional protein
Blunt and sticky ends
blunt= restriction enzyme cuts in the same positions in both sides
sticky= restriction enzymes does not cut in the same position
Suggest why the restriction enzyme has cut the human DNA in many places but has cut the plasmid DNA only once.
- enzymes only cut DNA at specific base sequence
- occurs once in plasmid and many times in human DNA;
How can a gene be expressed?
once a DNA fragment is isolated, it is possible to add a ‘promoter region’ which then allows the gene to be expressed once inserted into the bacterial plasmid.
Similarly, ‘a terminator region’ is also added to the fragment which stops transcription.
Explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected.
- isolate TARGET gene from organism
- using restriction endonuclease to get DNA
- produce sticky ends;
- use DNA ligase to join TARGET gene to plasmid;
- also include marker gene;
- add plasmid to bacteria to grow
- bacteria colonies not killed have antibiotic resistance gene and the TARGET gene;
Isolation using the gene machine
- Desired nucleotide sequence fed into a computer
- Synthesis of oligonucleotides (short sequences of nucleotides)
- Oligonucleotides are overlapped then joined together and made double stranded using the polymerase chain reaction (PCR)
- Gene is inserted into a bacterial plasmid
Advantages of the gene machine
- easily transcribed and translated by prokaryotes, as they have no introns in DNA
- faster (all enzyme catalysed reactions)
- more accurate
Insertion of genes into a vector
- Isolated Target DNA fragment inserted into vector DNA by cutting open the vector DNA using the SAME restriction endonuclease that was used to isolate DNA fragment.
- Produces complementary ‘sticky ends’ between the ends of DNA fragment and cut ends of vector DNA.
- Target DNA fragment anneals to vector DNA by complementary base pairing between their ‘sticky ends’.
- DNA ligase used to join the DNA fragment and vector DNA at the sugar phosphate backbone. (ligation) forms phosphodiester bonds.
- forms recombinant DNA.
Vector definition
A vector is a DNA carrier (i.e., bacterial plasmid or virus) used to transfer foreign DNA into cells.
Transformation producing recombinant organisms
process by which recombinant DNA VECTOR is transferred into a host cell (Bacteria)
Host cells which take up recombinant DNA are referred to as recombinant organisms or transformed organisms.
Plasmid vectors
circular pieces of DNA found in bacterial cells.
If bacterial cells are placed in solution with recombinant plasmids, they can be encouraged to take up plasmids from the solution under certain conditions
Describe a plasmid
- circular DNA;
- separate from main bacterial DNA;
- contains only a few genes
Outline a method for in vivo gene cloning
- Cut desired gene from desired organism;
- Using restriction endonuclease
1 using DNA polymerase;
2 cut plasmid open;
3 with same restriction endonuclease;
4 ref. sticky ends
5 use DNA ligase to join;
6 return plasmid to bacterial cells
Identifying transformed host cells
Transformed cells / organisms must be identified because:
1: some vectors take up target DNA to become recombinant
2: some host cells become transformed, by taking up recombinant vectors.
Only transformed host cells, which contain the ‘target gene’, will synthesise desired protein
Marker gene definition
allows easy identification of cells that have taken up a genetically transformed plasmid
Identifying transformed bacteria using antibiotic resistance genes
- Some cells will not have taken up any plasmid at all. These cells are killed by both types of antibiotic.
- Some cells will have taken up an “original” plasmid. These are resistant to both types of antibiotics.
- Some cells will have taken up a “transformed” plasmid. These cells are resistant to one type of antibiotic, but not the second (because the 2nd gene has been cut and disrupted by inserting foreign DNA).
The colonies of bacteria that survive the 1st antibiotic and are destroyed by the 2nd antibiotic are the cells you want.
Cloning (in vivo via bacteria)
Once you have identified the transformed bacterial cells then you can culture these bacteria as they reproduce by binary fission to produce genetically identical cells with the desired ‘inserted’ gene.
These organisms will produce large quantities of the desired protein
Cloning (in vitro via PCR)
Polymerase chain reaction (PCR) is used to AMPLIFY (make millions of copies of a single fragment of DNA).
The number of DNA molecules doubles with every cycle, making it rapid & efficient.
Describe the process of PCR
- Heat DNA to 95C which breaks (weak) hydrogen bonds
- Add primers and Add nucleotides;
- Cool to 50C to allow binding of nucleotides
- Add thermo stable DNA polymerase;
- Heat to 75C
- DNA polymerase joins nucleotides together;
- Repeat cycle many times;
Primers
are short pieces of single stranded DNA with complementary base sequences to bases at start of DNA fragment you want to isolate.
Primers also prevent DNA strands sticking back together and allows DNA polymerase to attach and join nucleotides together
Join to 5’ end
Why is the DNA heat to 95°C during PCR?
- Produce single stranded DNA
- Breaks WEAK hydrogen bonds between strands
Why do you add primers during PCR?
- Attaches to start of the gene
- Replication of base sequence from here;
- Prevents strands annealing
Calculating number of DNA strands after x number of cycles
2^n
Calculating number of cycles from number of DNA strands
Log2(number of DNA molecules)
Comparison between ‘in vitro’ and ‘in vivo’ DNA fragment cloning
Vivo- Can be used to produce protein or mRNA from the inserted DNA as well as the target DNA.
Vitro- Can only be used to copy DNA.
Vivo- slower
Vitro- quicker
Vivo- Modified DNA
Vitro- DNA not modified
PCR cycle graph
start= slow, DNA doubling each cycle
middle= exponential phase, going up in high numbers
end= plateau, limiting factor
- conc. primers = less new DNA strands
- conc. nucleotides = fewer nucleotides
Suggest one reason why DNA replication stops in the polymerase chain reaction
- Limited number of primers/nucleotides;
- DNA polymerase denatures
Explain why ‘base-pairs’ is a suitable unit for measuring the length of a piece of DNA.
- DNA = 2 chains
- Bases are a constant distance apart
- each base-pair is same length
Benefits of recombinant DNA technology
Develop medical applications:
-produce large quantities of insulin
Develop agricultural applications.
- Produce crops that are resistant to disease and/or extreme weather
Concerns regarding recombinant DNA technology
- Antibiotic resistance may spread owing to its use as a selection marker
- Inserting new genes into a crop plant could disrupt other gene/s function creating toxic products within genetically modified food sources
Suggest how a scientist would arrive at their hypothesis
looked for info on crop plants that grow at ↑ temp, that contain GB
assumed making plants produce GB makes them resistant to ↑ temp
Gene therapy using viruses as vectors
Viral DNA is cut using the same restriction endonuclease and joined to foreign DNA using DNA ligase. The virus then acts as a vector (DNA carrier).
Viruses can cause an immune response and the formation of cytotoxic T cells and memory B cells.
vector viruses are further modified to evade the immune response.
Using liposomes as vectors
Liposomes are lipid droplets which can cross the phospholipid bilayer and releases target DNA into the cell.
However, the DNA does not move into the nucleus and so does integrate into the genome of stem cells lining the airways, so new daughter cells will not have the functional gene. From this DNA, small amounts of mRNA can be produced
Somatic gene therapy
DNA transfer to our normal body tissue
Somatic limitations
- Not all cells take up new DNA
- Not all cells express DNA allele
- Only some tissue types are accessible i.e., lungs, needs to be repeated – as cells die replaced by cells with faulty DNA,
- Multiple treatments may be needed
- Body can produce an immune response to the vector
Germ line gene therapy
DNA transfer to cells that produce eggs or sperm
Germ line limitations
- effects of gene transfer are unpredictable and, even if the target disease was cured, further defects could be introduced into the embryo.
- Denial of human rights. Individuals resulting from germ line gene therapy would have no say in whether their genetic material should have been modified.
- Potential abuse. Germ line gene therapy could be used not only to eliminate disease, but also to enhance favourable characteristics and suppress unfavourable ones
Genetic screening
- sequence of nucleotides on mutated gene determined by DNA sequencing
- fragment of DNA with complementary bases, to mutant allele of gene, produced
- DNA probe formed by fluorescently labelling DNA fragment
- PCR used to produce multiple copies of DNA probe
- Probe added to single stranded DNA fragments from person being screened
- if complementary fragments present, DNA probe taken up and dye will fluoresce. if complementary fragment not present, DNA probe will not fluoresce
-When the DNA PROBE binds to its complementary target DNA, this is called DNA HYDRIDIZATION
What does the presence of fluorescence conclude?
- Carrier of a recessive allele known to cause a genetic disorder
- Contains mutations in known genes linked to increased risk of disease
- Contains genes that code for proteins that predict positive or negative response to a drug dosage
Gene probe definition
A short, single stranded DNA molecule with a complementary base sequence to DNA fragment
What are the regions between genes called?
VNTR’s –VARIABLE NUMBER TANDEM REPEATS
VNTR
Each individual has two copies of each VNTR one on each homologous chromosome. One copy inherited from each parent.
Differences in the length of VNTRs can be analysed via GEL ELECTROPHORESIS
Describe how genetic fingerprinting is carried out
- DNA extracted from sample;
- DNA hydrolysed into segments using restriction endonuclease;
- Must leave VNTR’s intact;
- DNA fragments separated using electrophoresis;
- mixture put into wells on gel and electric
current passed through - Immerse gel in alkaline solution so two strands of DNA separated;
- Probe added
- areas with probe identified using autoradiography
Name one mutagenic agent.
- high energy radiation
- benzene
- x rays
- uv (light);
A deletion mutation occurs in gene 1.
Describe how a deletion mutation alters the structure of a gene.
- removal of one or more bases
- frameshift
Describe how the bacteria containing the insulin gene are used to obtain sufficient insulin for commercial use
- use of fermenters;
- provides nutrients plus suitable conditions for optimum growth
- environmental factor;
- reproduction of bacteria;
- insulin accumulates and is extracted;
mRNA may be described as a polymer. Explain why.
- Made up of many similar molecules
Name three techniques used by scientists to compare DNA sequences.
- Polymerase Chain Reaction
- DNA fingerprinting
- Gel electrophoresis
