Manipulating Genomes

These flashcards cover key concepts in genome manipulation, DNA sequencing, genetic engineering, and bioinformatics as discussed in the provided lecture transcript.

What is DNA sequencing and what is its primary goal?

It is the process of determining the exact nucleotide base sequence (the order of A, T, C, and G) of an organism's genetic material.

Who developed the chain termination method for DNA sequencing?

Frederick Sanger and his team developed this technique, now known as Sanger sequencing.

What was the significance of the phiX174 virus in the history of genetics?

It was the first entire genome to be sequenced by Frederick Sanger's team in 1977.

Explain the role of dideoxynucleotides (ddNTPs) in the Sanger method.

They act as chain terminators. Because they lack a 3' hydroxyl group, DNA polymerase cannot form a phosphodiester bond with the next nucleotide, halting elongation.

How did the transition from radioactive labels to fluorescent tags advance DNA sequencing?

It reduced manual effort, increased safety, and allowed for automation, as lasers can detect the different colors associated with each base in a single reaction tube.

Define 'high-throughput sequencing'.

Modern techniques that allow the simultaneous sequencing of billions of DNA strands rapidly and at a significantly lower cost than traditional methods.

What is 'massively parallel sequencing'?

A high-throughput method that allows for the simultaneous replication and imaging of millions of DNA fragments on a flow cell.

What is the role of Bioinformatics in modern genetics?

It involves the development of software and computational tools to organize, store, and analyze large-scale biological data, such as whole-genome sequences.

How does Computational Biology differ from Bioinformatics?

Bioinformatics focuses on the data management and analysis, while computational biology uses that data to build theoretical models of biological systems.

Define Genomics.

The branch of molecular biology concerned with the structure, function, evolution, and mapping of genomes.

How is genome sequencing used to study evolutionary relationships?

By comparing DNA sequences between species, scientists can determine how recently they shared a common ancestor using the 'molecular clock' concept.

What are Variable Number Tandem Repeats (VNTRs)?

Short sequences of DNA in non-coding regions that repeat a variable number of times. The pattern is unique to individuals (except identical twins).

What is the primary purpose of DNA profiling?

To identify individuals or determine genetic relationships by looking at specific variable regions of the genome (VNTRs).

List the five stages of producing a DNA profile.

1. Extract DNA from the sample.
2. Digest the sample using restriction endonucleases.
3. Separate fragments via gel electrophoresis.
4. Hybridize with radioactive or fluorescent probes.
5. Visualize the evidence (e.g., via UV light or X-ray).

Explain the 'Digestion' stage of DNA profiling.

Restriction endonucleases are used to cut the DNA at specific recognition sites, leaving the repeated VNTR sequences intact within the resulting fragments.

What is the purpose of the Polymerase Chain Reaction (PCR)?

To exponentially amplify a small sample of DNA into billions of copies for analysis.

Describe the Denaturation stage of PCR.

The reaction mixture is heated to approximately 95^{\circ}C. This breaks the hydrogen bonds between complementary bases, separating the double-stranded DNA into single strands.

Describe the Annealing stage of PCR.

The temperature is lowered to approximately 55^{\circ}C. This allows DNA primers to bind (anneal) to the complementary sequences at the ends of the target DNA region.

Describe the Elongation/Extension stage of PCR.

The reaction is heated to 72^{\circ}C. Taq polymerase adds free nucleotides to the primers in the 5' to 3' direction to synthesize new DNA strands.

Why is Taq polymerase used in PCR instead of human DNA polymerase?

It is derived from the thermophilic bacterium Thermus\ aquaticus. It is heat-stable and does not denature at the high temperatures (95^{\circ}C) required to separate DNA strands.

What is the function of a 'Primer' in PCR?

A short, single-stranded sequence of DNA that provides a starting point for DNA polymerase, as the enzyme can only add nucleotides to an existing chain.

How does Gel Electrophoresis separate nucleic acids?

Fragments are placed in an agarose gel and an electric current is applied. Negatively charged DNA (due to phosphate groups) moves toward the positive anode; smaller fragments move faster and further.

What materials are needed for Gel Electrophoresis besides the DNA and gel?

A buffer solution (to maintain pH and conduct electricity) and a loading dye (to help track the migration of the DNA).

Define Restriction Endonucleases.

Enzymes that cut DNA at specific palindromic sequences called recognition sites.

Distinguish between 'Sticky Ends' and 'Blunt Ends'.

Sticky ends have staggered cuts with overhanging single-stranded bases that join easily to complementary DNA; blunt ends are cut straight across both strands.

What is the role of DNA Ligase in genetic engineering?

It catalyzes the formation of phosphodiester bonds between the sugar-phosphate backbones of DNA fragments, effectively 'gluing' them together.

How is Reverse Transcriptase used to isolate genes?

It uses an mRNA template to synthesize a complementary DNA (cDNA) strand. This is useful because mRNA has already had non-coding introns removed.

Define 'Vector' in the context of genetic engineering.

A DNA molecule (like a plasmid or bacteriophage) used to transport a foreign gene into a host cell.

What is 'Transformation'?

A technique where bacterial cells take up foreign DNA from their surroundings, often induced by heat shock and calcium chloride treatment.

Explain 'Electroporation'.

The use of high-voltage electrical pulses to create temporary pores in the cell membrane, allowing large molecules like recombinant plasmids to enter.

How are marker genes used to identify successfully transformed bacteria?

Markers like antibiotic resistance genes allow only the bacteria that have successfully taken up the plasmid to grow on media containing the antibiotic.

What is 'Insertional Inactivation'?

When the target gene is inserted directly into a marker gene (e.g., fluorescence), 'breaking' that gene's function. This helps identify recombinant colonies by their lack of that trait.

Define Synthetic Biology.

An interdisciplinary field that redesigns existing natural biological systems or creates entirely new biological parts, pathways, and organisms.

What is DNA Barcoding and how is it used?

An identification method using short, standardized sections of DNA from specific genes (like cytochrome c oxidase I) to differentiate between species.

Explain 'Somatic Cell Gene Therapy'.

The insertion of a functional gene into differentiated body cells of a patient. The effect is temporary (cells die) and is NOT passed on to offspring.

Explain 'Germ Line Cell Gene Therapy'.

The insertion of a functional gene into gametes or early zygotes. This results in the gene being present in every cell of the offspring and being passed to future generations.

What are three ethical concerns regarding Genetically Modified (GM) crops?

1. Environmental: Creation of superweeds through cross-pollination.
2. Ecological: Negative impact on non-target species and biodiversity.
3. Economic: Patents making seeds expensive for farmers in developing nations.

What ethical concern exists regarding the use of modified organisms in warfare?

The potential to use synthetic biology to design highly virulent or antibiotic-resistant pathogens for biological warfare.

How does sequencing pathogens assist in medical management?

It allows scientists to track outbreaks, identify the source of infections, and monitor mutations that might cause drug resistance.

Define the significance of palindromic recognition sites.

These are sequences that read the same in the 5' to 3' direction on both strands, allowing restriction enzymes to cut both strands at specific points.

Why is the use of a DNA probe necessary in DNA profiling?

Probes are labeled (fluorescently or radioactively) and complementary to the VNTRs, allowing researchers to see specific bands on the profile.