Topic 8 - Gene Technologies Flashcards
What is DNA ligase?
An enzyme that joins together fragments of newly synthesised DNA to form a seamless strand
What is gene cloning?
Makes indentical copes of a piece of DNA, such as a gene
What is a gene marker?
A gene that is transferred with the desired gene to enable scientists to identify which cells have been successfully altered and now contain recombinant DNA.
What is gene therapy?
Using various mechanisms to alter a person’s genetic material to treat or cure diseases.
What is genetic modification?
Organisms that have had their DNA altered through combinant DNA technology.
What is In Vivo cloning?
Involves the insertion of a DNA sequence into a host organism where it is then allowed to replicate.
What is a plasmid?
Small circular rings of double stranded DNA
What are the 4 types of gene technology?
1) DNA profiling
2) Genomic sequencing
3) Genetic engineering
4) Gene therapy
What is recombinant DNA?
Where fragments of foreign DNA are inserted into other sections of DNA
What is a vector?
A DNA molecule that is used as a vehicle to carry a particular DNA segment into a host cell as a part of a cloning or recombinant DNA technique
What is a proteome?
All the proteins produced by the genome
What are the 5 main steps in making proteins?
1) Isolation - desired protein in DNA fragements
2) Insertion - DNA fragment into a vector
3) Transformation - DNA into a suitable host cell
4) Identification - host cells that have successfully taken up the gene
5) Growth/cloning - population of host cells
What is a transgenic organism?
Organisms that have had DNA from another individual, often from another species, inserted into their genome. Contain recombinant DNA.
What is a retrovirus?
Virus that contains reverse transcriptase
How is reverse transcriptase used to isolate a gene?
1) Cut out a DNA fragment containing a desired gene from a cells genome.
2) Make a required gene from a cells mRNA, using reverse transcriptase.
3) Make artificial genes by working backwards from the protein or the mRNA, that is use a gene machine to produce a specific protein if we know the protein we want and its amino acid sequence.
How is reverse transcriptase used to make DNA from RNA?
Reverse transcriptase is used to make DNA from RNA. This DNA is known as complementary DNA (cDNA) because it is made up of the nucleotides that are complementary to the mRNA.
How is a gene inserted into a plasmid vector?
1) There is a DNA from cell which manufactures the desired protein and a plasmind from a bacterium.
2) DNA and plasmid from bacterium are both cut using the same restriction endonuclease.
3) The sticky ends on the DNA fragments are revealed.
4) DNA fragment and open plasmid are mixed together with DNA ligase.
5) The plasmid with the new gene incorporated is formed.
What is bacterial conjugation?
Microorganisms can naturally exchange genetic material in a process called conjugation. Genetic material in the form of plasmids can be copied and passed between bacteria.
How do viruses act as vectors?
Viruses naturally transfer their genetic material into their host’s cells, as they need the host to produce viral proteins to allow them to replicate. Scientists use viruses such as the adenovirus as vectors in DNA technology.
How is cDNA formed?
1) A few extra nucleotides are added to create sticky ends.
2) A complete cDNA is sealed
What is genetic engineering?
Genetic engineering is the deliberate manipulation of genetic material to modify an organism’s characteristics, often involving gene transfer.
What is recombinant DNA?
Recombinant DNA is DNA that is altered to contain nucleotides from two different organisms. It allows DNA fragments to be transferred between organisms. Organisms that receive transferred DNA fragments are called genetically modified or transgenic organisms.
What are the 6 key stages in gene transfer?
1) The desired gene is identified and isolated.
2) Multiple copies of the gene are made using the polymerase chain reaction (PCR).
3) The gene is inserted into a vector.
4) The vector delivers the gene into cells in a different organism.
5) Cells with the new gene are identified, such as by using marker genes.
6) Cells with the new gene are cloned.
What are the 3 main methods used to produce DNA fragments to transfer?
1) Making complementary DNA (cDNA) using reverse transcriptase and mRNA.
2) Cleaving DNA from a donor organism with restriction enzymes.
3) Synthesising new custom DNA sequences from nucleotides using a gene machine.
How does reverse transcriptase produce DNA fragments?
1) mRNA is extracted from cells
2) mRNA is reverse transcribed using the reverse transcriptase enzyme and DNA nucleotides
3) This makes a cDNA strand identical to the original DNA strand, and cDNA is isolated from the mRNA strand
4) cDNA, free nucleotides, and DNA polymerase can then be used to form the other strand of DNA, reforming the desired gene
How are restriction enzymes used to cut DNA?
1) DNA is incubated with chosen restriction enzyme(s).
2) Restriction enzymes identify palindromic sequences in the DNA double helix and cut double-stranded DNA if their recognition sequence is present
3) Recognition sequences at either end of a desired DNA fragment allow restriction enzymes to separate the fragment from the rest of the DNA to obtain the desired gene
4) Enzymes cut target gene fragment out via hydrolysis reaction
5) Different restriction enzymes cut at different sequences based on their active site shape
What are ‘sticky ends’?
The cut DNA often has sticky ends. These are short overhanging sequences of unpaired bases that can bind to other DNA fragments when they are inserted into vectors.
How can new custom DNA be synthesised?
Gene machines (DNA synthesisers) can now synthesise completely new DNA sequences.
1) Choose codons for the desired amino acid sequence from a known protein structure.
2) Use a computer to direct the synthesis of short fragments of DNA (oligonucleotides) in gene machines.
3) Join the fragments together to make a longer sequence of nucleotides, forming the desired gene.
4) Polymerase chain reaction (PCR) constructs a complementary DNA strand and amplifies the gene to produce multiple copies.
What are the 3 stages of In Vivo Cloning?
Step 1) Inserting DNA fragments into vectors.
Step 2) Transferring recombinant DNA into host cells.
Step 3) Identifying transformed host cells.
How are DNA fragments inserted into vectors?
1) A vector is cut open at a specific site using a restriction enzyme, creating sticky ends.
2) The same restriction enzyme is used to cut the target DNA fragment, creating complementary sticky ends.
3) DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the two strands of DNA, joining the sticky ends of the vector and DNA fragment together.
4) The newly formed combined DNA molecule is known as recombinant DNA.
How is recombinant DNA trasferred into host cells?
Transformation involves introducing vectors with recombinant DNA into host cells, transforming these cells. The vectors are usually either plasmids or bacteriophages.
Plasmid vectors:
- These are small, circular DNA molecules, typically found in bacteria.
- Host cells are treated to enhance the uptake of plasmids that have recombinant DNA.
- For instance, applying calcium ions and temperature shifts can make bacterial membranes more permeable to plasmids.
Bacteriophage vectors:
- These are viruses that infect bacteria.
- Bacteriophages inject their DNA into host bacterial cells during infection.
- The phage DNA, now carrying the recombinant DNA, inserts into the host’s DNA.
How are transformed host cells identified?
As only around 5% of host cells may uptake DNA, it’s important to identify which cells have been successfully transformed. Marker genes indicate which host cells took up recombinant DNA:
- They are inserted into vectors alongside target genes.
- Transformed cells are cultivated on selective agar plates.
- Only transformed cells display the characteristics encoded by marker genes.
- These transformed cells can then be cultured to mass-produce the target DNA fragment through cellular replication.
What are the different types of marker genes?
Different marker genes can identify which host cells took up recombinant DNA. Some examples of marker genes include:
- A marker gene for a specific trait, like antibiotic resistance, ensures that only transformed cells form colonies.
- A marker gene that is visible under UV light like green fluorescent protein (GFP).
- Inserting a marker gene within the GFP gene inhibits fluorescence if it is successfully incorporated.
- A marker gene coding for an enzyme that alters the colour of a specific substrate.
What is a promotor region?
Provide a binding area for RNA polymerase, signalling where the transcription of the gene should start.
What is a terminator region?
Indicate where transcription should end, releasing RNA polymerase and terminating transcription.
What is the polymerase chain reaction (PCR)?
The polymerase chain reaction (PCR) is a method for amplifying DNA fragments rapidly and efficiently. ‘Amplifying’ means that a large number of DNA fragments are being produced.PCR is an automated technique that does not require living cells to rapidly replicate specific DNA fragments. It is sometimes called ‘in vitro cloning’.
What are the components needed for PCR?
- DNA fragment - This is the specific target DNA template sequence that needs to be replicated.
- Primers - These are short sequences of nucleotides that attach to the start and end of the DNA fragment to be copied.
- DNA polymerase - This is an enzyme that creates new DNA strands by adding DNA nucleotides.
- Free nucleotides - The building blocks (A, T, C, and G) that are used to construct the new DNA strands.
- Thermocycler - This is a device that precisely heats and cools the PCR mixture to facilitate the reaction.
What are the 3 stages of the polymerase chain reaction?
1) Seperation (denaturation) - 95 degrees - Heating the DNA seperates the hydrogen bonds between its two complementary strands.
2) Addition of primers (annealing) - 55 degrees - The primers attach to the specific starting points on each of the seperated DNA strands forming hydrogen bonds.
3) DNA synthesis (extension) - 72 degrees - DNA polymerase adds free nucleotides to the ends of the primers, extending the DNA strand to form a complete copy.
What are the advantageous of PCR?
- Rapid speed - It can produce approximately 100 billion copies in just a few hours, which is not possible with in vivo cloning.
- Precision - The automated nature of thermocycling ensures accurate amplification of the DNA fragment.
- Low DNA needs - Even tiny samples can be amplified to produce a large quantity for analysis.
- No cells needed - This method is simpler and more straightforward than traditional in vivo techniques, which are typically slower and more complex.
What is DNA Probe?
A DNA probe is a short, labelled, single-stranded DNA sequence that can identify a specific allele. It does this by binding to a complementary DNA sequence. For example, a DNA probe can determine whether an individual carries a mutated allele responsible for a genetic disorder.
How is DNA screened for a specific allele using a DNA probe?
- Denaturation - An individual’s DNA strands are heated causing them to separate into two single strands.
- Fragmentation - Digest the DNA sample into fragments using restriction enzymes.
- Mixing - Add fluorescently or radioactively labelled DNA probes.
- Hybridisation - DNA probes bind with the target sequence.
- Identification - Visualise any DNA probe that has hybridised to DNA fragments under UV light (for fluorescently labelled DNA probes) or X-ray (for radioactively labelled probes) to signal whether the allele is present.
How can gene therapy correct genetic disorders?
1) Silencing defective dominant alleles that could lead to dominant genetic disorders.
2) Adding a working dominant allele to silence defective recessive alleles that could cause recessive genetic disorders.
3) Using vectors such as viruses or liposomes to transport healthy alleles into cells.
4) Focusing on somatic cells for treating individuals or germline cells to prevent passing the disease to offspring.
What is gel electrophoresis?
Gel electrophoresis is a technique used to separate molecules such as DNA, RNA, or proteins based on size by using an electric current applied to an agarose gel matrix.
How is a gel electrophoresis set up?
1) Insert a gel tray with solidified agarose gel into a gel tank.
2) Ensure the wells are close to the negative electrode to position the gel correctly.
3) Pour a buffer solution over the gel until it is submerged to maintain a constant, suitable pH throughout the experiment.
How are the samples loaded into the wells in gel electrophoresis?
1) Mix the DNA samples with loading dye to make them visible.
2) Carefully deposit equal volumes of each sample into the wells using a micropipette.
3) Touch the micropipette tip to the buffer, not the bottom of the gel, to prevent damaging the gel.
4) Keep a record of which sample is in each well for later analysis.
What happens during gel electrophoresis?
1) A voltage (around 100 V) is applied across the gel.
2) Fragments of DNA move towards the positive electrode (anode).
3) The smaller fragments travel faster and thus separate by size.
4) Continue the process for about 30 minutes or until the dye approaches the end of the gel.
How are the results visualised in gel electrophoresis?
1) Switch off the voltage and remove the gel from the tank.
2) Apply a stain to the DNA to reveal the bands of fragments.
3) Assess the migration distances of the bands to approximate the sizes of the fragments.
What is genetic fingerprinting?
Genetic fingerprinting, sometimes called DNA profiling, is a technique used to identify unique DNA patterns in individuals, to help identify individuals in forensics or family relationships.
What does genetic fingerprinting rely on?
Genetic fingerprinting relies on the fact that, with the exception of identical twins, every person’s DNA sequence is distinct. This is due to variation in the sequence and length of unique non-coding, repetitive DNA segments called variable number tandem repeats (VNTRs, sometimes called minisatellites).
What are the key features of variable number tandem repeats (VNTRs)?
- They are present across the genomes of most eukaryotes.
- They are not involved in protein coding.
- They have extensive variability in sequence and length among individuals.
- Their length and location are heritable.
- The chance of two individuals who are not identical twins sharing identical VNTR patterns is very low.
- A high similarity in VNTR patterns indicates two individuals may be closely related.
What are STRs?
Shorttandem repeats (STRs, sometimes called microsatellites) are repeated sequences of nucleotides that are smaller than VNTRs and can also be used in genetic fingerprinting.
How is DNA profile created?
1) DNA extraction - DNA is extracted from a tissue sample and amplified using PCR.
2) DNA digestion - Restriction enzymes are used to cut the DNA into fragments at points near the VNTR sequences.
3) Fragment separation - Gel electrophoresis separates the fragments by size, and they are denatured to produce single strands.
4) Hybridisation - Specific radioactive or fluorescent probes bind to complementary VNTR sequences.
5) Development - The positions of the probes are revealed, creating a barcode-like pattern of DNA bands unique to each individual.
What are some uses of genetic fingerprinting?
- Establishing paternity by matching bands on a child’s genetic fingerprint to those of potential parents and assessing the similarities between VNTR patterns.
- Identifying suspects from crime scene DNA.
- Supporting criminal convictions with match probability calculations.
- Identifying the risk of genetic disorders and predicting their onset and severity.
What are some limitations of genetic fingerprinting?
- Environmental contamination may compromise results.
- Close genetic relatives could have similar fingerprints.
- Assumptions about genetic variation in populations underpin probability calculations, meaning genetic fingerprinting does not prove guilt or causation and other evidence must also be taken into account.
