T21 Flashcards
How are DNA fragments transferred between organisms?
The desired gene is identified and isolated.
Multiple copies of the gene are made using the polymerase chain reaction (PCR).
The gene is inserted into a vector.
The vector delivers the gene into cells in a different organism.
Cells with the new gene are identified, such as by using marker genes.
Cells with the new gene are cloned.
How can reverse transcriptase produce DNA fragments?
mRNA is extracted from cells.
mRNA is reverse transcribed using the reverse transcriptase enzyme and DNA nucleotides.
This makes a cDNA strand identical to the original DNA strand, and cDNA is isolated from the mRNA strand.
cDNA, free nucleotides, and DNA polymerase can then be used to form the other strand of DNA, reforming the desired gene.
How are restriction endonucleases used to cut DNA?
DNA is incubated with chosen restriction enzyme(s).
Restriction enzymes identify palindromic sequences in the DNA double helix and cut double-stranded DNA if their recognition sequence is present.
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.
Enzymes cut target gene fragment out via hydrolysis reaction.
Different restriction enzymes cut at different sequences based on their active site shape.
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 using a gene machine/DNA synthesiser?
Choose codons for the desired amino acid sequence from a known protein structure.
Use a computer to direct the synthesis of short fragments of DNA (oligonucleotides) in gene machines.
Join the fragments together to make a longer sequence of nucleotides, forming the desired gene.
Polymerase chain reaction (PCR) constructs a complementary DNA strand and amplifies the gene to produce multiple copies.
Describe the steps in in vivo cloning
Step 1 - Inserting DNA fragments into vectors
A vector is cut open at a specific site using a restriction enzyme, creating sticky ends.
The same restriction enzyme is used to cut the target DNA fragment, creating complementary sticky ends.
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.
The newly formed combined DNA molecule is known as recombinant DNA.
Step 2 - Transferring recombinant DNA 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.
Step 3 - Identifying transformed host cells
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.
Different types of marker genes
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.
Adding promoter and terminator regions
For the protein coded by an inserted DNA fragment to be produced, vectors require certain control elements.
Control elements that a vector needs:
Promoter regions - Provide a binding area for RNA polymerase, signalling where the transcription of the gene should start.
Terminator regions - Indicate where transcription should end, releasing RNA polymerase and terminating transcription.
Describe the steps in in vitro cloning
- Separation (denaturation)
95°C
Heating the DNA separates the hydrogen bonds between its two complementary strands. - Addition of primers (annealing)
55°C
The primers attach to the specific starting points on each of the separated DNA strands by forming hydrogen bonds. - DNA synthesis (extension)
72°C
DNA polymerase adds free nucleotides to the ends of the primers, extending the DNA strand to form a complete copy.
Free deoxynucleotide triphosphate molecules (dNTPs) provide the energy for the formation of phosphodiester bonds.
What are the advantages 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 a DNA probe?
A short, labelled, single-stranded DNA sequence that can identify a specific allele.
How are DNA probes created?
The target allele’s base sequence is first identified using DNA sequencing so that a complementary probe can be made. Then, the sequence is replicated using PCR.
What is the process of screening DNA 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 do DNA microarrays work?
Extract DNA from the species or cells of interest.
Fragment and fluorescently label these DNA samples.
Mix the labelled DNA and wash it over the microarray probes.
DNA fragments that match any of the probes will hybridise.
The array is then viewed under UV light.
Any fluorescent spots show that the sample contains those fragments.
How does gene therapy seek to correct genetic disorders?
Silencing defective dominant alleles that could lead to dominant genetic disorders.
Adding a working dominant allele to silence defective recessive alleles that could cause recessive genetic disorders.
Using vectors such as viruses or liposomes to transport healthy alleles into cells.
Focusing on somatic cells for treating individuals or germline cells to prevent passing the disease to offspring.
What is gel electrophoresis and how is it set up?
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 to set up gel electrophoresis:
Insert a gel tray with solidified agarose gel into a gel tank.
Ensure the wells are close to the negative electrode to position the gel correctly.
Pour a buffer solution over the gel until it is submerged to maintain a constant, suitable pH throughout the experiment.
How do you run electrophoresis
To load samples:
Mix the DNA samples with loading dye to make them visible.
Carefully deposit equal volumes of each sample into the wells using a micropipette.
Touch the micropipette tip to the buffer, not the bottom of the gel, to prevent damaging the gel.
Keep a record of which sample is in each well for later analysis.
DNA molecules carry a negative charge due to their phosphate groups.
During electrophoresis:
A voltage (around 100 V) is applied across the gel.
Fragments of DNA move towards the positive electrode (anode).
The smaller fragments travel faster and thus separate by size.
Continue the process for about 30 minutes or until the dye approaches the end of the gel.
The size-based separation occurs because the gel’s mesh matrix slows down larger fragments more than the smaller ones. The pore size of the gel affects the rate at which fragments move.
How to visualise results from electrophoresis
Switch off the voltage and remove the gel from the tank.
Apply a stain to the DNA to reveal the bands of fragments.
Assess the migration distances of the bands to approximate the sizes of the fragments.
What are key features of 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.
How to create a genetic fingerprint?
DNA extraction - DNA is extracted from a tissue sample and amplified using PCR.
DNA digestion - Restriction enzymes are used to cut the DNA into fragments at points near the VNTR sequences.
Fragment separation - Gel electrophoresis separates the fragments by size, and they are denatured to produce single strands.
Hybridisation - Specific radioactive or fluorescent probes bind to complementary VNTR sequences.
Development - The positions of the probes are revealed, creating a barcode-like pattern of DNA bands unique to each individual.
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.
