Gene Technology Flashcards
State the three methods for obtaining DNA fragments
Using restriction endonuclease
Using reverse transcriptase
Using a ‘gene machine’
Describe how restriction endonuclease can obtain DNA fragments
Group of enzymes that n aurally occur in bacteria
They cut DNA at a specific sequence of bases (called a recognition sequence) by hydrolysing the phosphodiester bonds in the sugar phosphate backbone
They cut the DNA between opposite base pairs in a staggered structure. This creates sticky ends that can bind to other sticky ends with complimentary base pairing
What are sticky ends?
Single stranded sections of DNA that form an overhang at the end of a double stranded molecule
How is a gene machine used to obtain fragments of DNA?
Where genes are synthesised artificially:
- the amino acid sequence of a gene is determined
- the mRNA codons for each amino acids are searched
- the complimentary DNA triplets for the mRNA sequences are worked out and the gene is produced
-the genes are checked using standard sequencing techniques. Any with errors are rejected
What are benefits of producing artificial genes with the gene machine?
Genes are free of introns and other non-functioning DNA- so they can be transcribed and translated
After isolation of fragments of DNA, how is the number of DNA fragments increased?
Polymerase Chain reaction
What are primers?
A short sequence of single stranded nucleotides, that have a specific base sequence
What are the components of a thermal cycle used in PCR?
DNA fragments that need to be copied
Taq polymerase
Free nucleotides
Primers
Explain the process of the polymerase chain reaction
In the thermal cycler, at 95*C the hydrogen bonds are broken and DNA double strand separates.
Temp is reduced to 55, primers attach to complimentary base sequences at the ends of the single strand
DNA polymerase attaches and the temp is raised to 72 (optimum temp for enzymes)
Free nucleotides attach to the single stranded templates via complimentary base pairing
DNA polymerase joins nucleotides together with phosphodiester bonds
DNA doubles in quantity
How can reverse transcriptase be used to obtain DNA fragments?
Viruses called retroviruses contain RNA but not DNA, therefore they use the enzyme reverse transcriptase turns RNA into DNA before replication, once inside living cells.
This property of revers transcriptase is used to convert mRNA to cDNA (complimentary DNA)
Reverse transcriptase and DNA nucleotides are added to mRNA
What are two advantages for using reverse transcriptase to produce a gene instead of removing DNA from an organisms genome?
mRNA has a higher concentration. Within cells so it is easier to isolate than genes
The cDNA that is produced will not contain non-coding introns, so the gene will be able to be expressed in prokaryotic cells
What is gel electrophoresis?
A technique that separates different pieces of DNA based on their length
Once gel eletrophoresis has separated pieces of DNA, what can they be used for?
Locating defective genes that are used against specific diseases, using gene probes
Analysing DNA fragments to produce a genetic fingerprint
Describe the process of gel electrophoresis
DNA samples are amplified with PCR, and cut into fragments with restriction enzymes
Fragments placed into wells at one end of a thin bit of gel
An electric current is passed through the gel, the DNA fragments are attracted to the positive electrode
Molecules move through the gel, the shorter the lengths log DNA, then faster and further they move in a given time
During gel electrophoresis, why are DNA fragments attracted to the positive electrodes?
DNA is negatively charged due to the phosphate ions
What is used to mark the DNA fragments during gel electrophoresis?
Stationed with fluorescent chemicals
Radioactively labelled, and the. An x-ray film is used to identify their positions
During gel electrophoresis, what are size markers and what are they used for?
A series of bands of DNA fragments of known sizes, that can be used to estimate the sizes of the unknown fragments by comparing the positions of their bands
What is genetic screening and diagnosis?
Where a gene probe is used to identify if a gene or allele is present in a DNA fragment, after gel electrophoresis
Explain what a gene probe is, and how it is used in genetic screening?
A gene probe is a single stranded short sequence of DNA nucleotides that have a complimentary base sequence to a specific gene or allele
The DNA from gel electrophoresis is made single stranded using an alkali.
The gene probe is added
The gene probe will bind to the target gene or allele if it is present in a DNA fragment, it will be labelled with a radioactive or fluorescent tag to it can be seen
What genes/ alleles are most commonly screened for in genetic screening?
Oncogenes that may cause cancer
Sickle cell anaemia
Cystic fibrosis
What is used to advise people on genetic screening?
Genetic counselling
What is genetic fingerprinting?
A diagnostic tool that analyses the small differences in DNS bade sequences between individuals of the same species, by looking at non-coding DNA instead of coding DNA
What DNA does genetic fingerprinting use and not use?
Does not use coding DNA
Does NOT use non-coding DNA found within genes (introns)
DOES use the non-coding DNA found between genes
What are variable number tandem repeat?
Repetitive sequences of non coding DNA found between genes, used in genetic fingerprinting
Each person has a unique pattern
Describe the technique used for genetic fingerprinting
Extraction- DNA is extracted from a nucleus, and amplified using PCR
Digestion- restriction endonuclease cut recognition sites close to the variable number tandem repeats
Separation- gel electrophoresis separates the DNA fragments, and double stranded DNA is turned into single stranded in alkali
Binding to DNA probes- DNA probes, that are radioactive or fluorescent, bind to the variable number tandem repeats. Different repeats will cause a different amount of DNA probes to bind
Visualisation- series of bands are seen by using the fluorescent and radioactive probes
DNA from crime scenes can then be compared to suspects DNA to see if the patterns are identical
What are the 4 uses of DNA fingerprinting?
Paternity tests- seeing if genetic relationships are seen
Forensic science- crime scenes
Medical diagnosis- sample of DNA can be compared with someone who has a disease to see if they are similar
Plant and animal breeding- prevent inbreeding by seeing how closely some animals of the same species are related
What is in vivo cloning?
The transfer of DNA fragments from one organism to another. The genetic code is universal, as well as transcription and translation being universal. Therefore the DNA can be translated within the organism
Describe the process of in vivo cloning
The DNA fragment is prepared for insertion
Insertion of the DNA fragment into a vector (which carries things between organisms)
Transformation- the vector transfers then DNA into suitable host cells
Identification of the transformed cells using gene markers
The transformed cells population grow and clone
In vivo cloning- how is DNA prepared for insertion?
A promoter sequence is added to the start of the DAN fragment, to provide a binding sit for RNA polymerase
A terminator sequence is added to the end, to release the RNA polymerase and end transcription
In vivo cloning- what is used to transfer the DNA fragments from organism to organism, using vectors?
A vector transports the DNA into a host cell, using recombinant plasmids.
The vectors contain recognition sits, so restriction enzymes can cut them open.
They contain ampicillin resistant genes and green fluorescent protein genes- which are used as gene markers
In vivo cloning- Describe the process of modifying plasmids to make recombinant plasmids
A restriction enzymes is used to cute the gene from the DNA fragments, this creates sticky ends
The same restriction enzyme cuts the plasmids to create complimentary sticky ends
Gene, plasmid and DNA ligand enzyme mixed in a test tube, gene and plasmid are joined by DNA ligand. Sticky ends are joined by phosphodiester bonds
This is a recombinant plasmid, ready for in vivo cloning
Sometimes the plasmid sticks ends rejoin without the gene
In vivo cloning- why will only a small number of bacteria cells be ‘transformed’?
Some bacteria cells will take in the original plasmids
Some bacteria will not take in any plasmids at all
In vivo cloning- what are marker genes used for and how do they work?
They differentiate between the bacterial cells that tackle in no plasmids, the original plasmids and the recombinated plasmids/
The green fluorescent protein (GFP) will only be expressed if a gene is inserted into the plasmid (in a recombinant plasmid). And so it will glow when it is exposed to UV light
What is gene therapy?
A treatment for genetic diseases. Where the genotype is altered by supplementing specific alleles
Describe the process of gene therapy
The non-defective allele is introduced into cells, so it is present with the defective allele:
It is first inserted into a vector, which delivers the allele into the nucleus of the cell
The cell transcribes the gene to mRNA
mRNA is translated into a functional protein, genetic disease is cured
What type of alleles does gene therapy work for, and why?
Recessive alleles only, because the defective alleles are still present, so if the original allele was dominant it would still be expressed even if a normal recessive allele is added.
Give examples of how genes can be modified in agriculture
Genes that are resistant to pests, diseases and herbicides can be added to plant species
Genes that develop a tolerance to extreme environmental conditions can be added to species
What are direct uses of genetically modified bacteria?
Used to increase the quantity of antibiotics and the rate in which they are made
To produce hormones such as insulin, oestrogen and testosterone
What are some arguments for recombinant DNA technology?
Microbes and other cells can be modified to produce useful substances such as insulin
GM crops can help to prevent diseases
Gene therapy can be used to treat genetic diseases such as cystic fibrosis
What are some arguments against recombinant DNA technology?
Cannot predict the risks when releasing GM organisms into the environment
Useful genes may mutate into harmful genes
Ethical issues- how far should we take gene technology?
