8.4 - Gene Technologies Flashcards

Question

How does reverse transcriptase produce DNA fragments?

Answer 1

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

Answer 2

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

Answer 3

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.

Answer 4

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.

Answer 5

Step 1) Inserting DNA fragments into vectors. Step 2) Transferring recombinant DNA into host cells. Step 3) Identifying transformed host cells.

Answer 6

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.

Answer 7

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.

Answer 8

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.

Answer 9

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.

Answer 10

Provide a binding area for RNA polymerase, signalling where the transcription of the gene should start.

Answer 11

Indicate where transcription should end, releasing RNA polymerase and terminating transcription.

Answer 12

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'.

Answer 13

- 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.

Answer 14

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.

Answer 15

- 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.

Answer 16

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.

Answer 17

- 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.

Answer 18

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.

Answer 19

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.

Answer 20

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.

Answer 21

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.

Answer 22

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.

Answer 23

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.

Answer 24

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.

Answer 25

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).

Answer 26

- 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.

Answer 27

Short tandem repeats (STRs, sometimes called microsatellites) are repeated sequences of nucleotides that are smaller than VNTRs and can also be used in genetic fingerprinting.

Answer 28

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.

Answer 29

- 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.

Answer 30

- 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.