Molecular Technologies

What is the purpose of PCR?

To make many copies of a specific stretch of DNA.

What is reverse transcriptase used for?

To use RNA as a template to make DNA.

What are the two main types of DNA molecules involved in gene cloning?

Chromosomal DNA and Vector DNA.

What does gene cloning allow researchers to do?

Locate and copy a specific gene from all the DNA present in an organism.

What is a vector in gene cloning?

A carrier for the DNA segment that is to be cloned.

What are restriction enzymes?

Enzymes that cut DNA into pieces, used in gene cloning.

What is the role of the lacZ gene in gene cloning?

To differentiate between colonies with a recircularized vector and those with a recombinant vector.

What is the significance of plasmids in gene cloning?

They often have selectable markers, such as antibiotic resistance genes.

What is the first step in preparing chromosomal DNA for cloning?

Obtain cellular tissue from the organism of interest.

What is the function of dNTPs in PCR?

They provide the precursors for DNA synthesis.

Who developed the Polymerase Chain Reaction (PCR)?

Kary Mullis in 1985.

What is required as starting material for PCR?

Template DNA, oligonucleotide primers, and dNTPs.

What is a recombinant vector?

A vector that has had chromosomal DNA inserted into it.

What is the typical length of oligonucleotide primers used in PCR?

About 15 to 20 nucleotides long.

What does gene cloning enable besides DNA sequencing?

Gene editing, DNA probes, and expression of cloned genes.

What is the process of extracting and purifying DNA from cells called?

Biochemical techniques.

What happens to the DNA carried by a vector when it replicates in a host cell?

The DNA that it carries is also replicated.

What is a key difference between gene cloning and CRISPR-Cas9?

CRISPR-Cas9 requires a fully sequenced genome.

What is the role of the origin of replication in vector DNA?

It determines whether a vector can replicate in a particular host cell.

What is the average score for Exam 2 mentioned in the lecture?

67%.

What is the purpose of using iClickers in the lecture?

To gather student feedback on exam preparation and expectations.

What is the significance of the term 'recombinant vector'?

It indicates that chromosomal DNA has been inserted into the vector.

What is the primary function of gene cloning?

To isolate and make many copies of a specific gene.

What are the two natural sources from which vectors are derived?

Plasmids and viruses.

How does PCR differ from traditional gene cloning?

PCR can copy DNA without the aid of vectors and host cells.

What is the main challenge of gene cloning mentioned in the notes?

It is time-intensive and only works for particular applications.

What is Taq polymerase?

A DNA polymerase isolated from the bacterium Thermus aquaticus, necessary for PCR due to its thermostability.

What are the three steps of a PCR cycle?

1. Denaturation - DNA strands are separated; 2. Primer annealing - Oligonucleotide primers bind to the DNA strands; 3. Primer extension - Nucleotides are added to the primers.

How many cycles are typically involved in a PCR run?

20 to 30 cycles.

What is the increase in target DNA sequence after 20 cycles of PCR?

220-fold (~1 million-fold).

What is Reverse Transcriptase PCR (RT-PCR) used for?

To detect and quantify the amount of RNA in living cells.

What is the first step in RT-PCR?

RNA is isolated from a sample.

What does Real-Time PCR measure?

The amount of a specific mRNA in a sample using fluorescence changes.

What happens during the exponential phase of Real-Time PCR?

The product doubles with every cycle until the Cycle Threshold (Ct) is reached.

What is the Dideoxy Method of DNA Sequencing?

A sequencing method that uses dideoxynucleotides to terminate DNA strand growth.

What is required before DNA sequencing?

The DNA to be sequenced must be obtained in large amounts, often using cloning or PCR.

What is the purpose of automated DNA sequencing?

To determine the sequence of DNA using fluorescently labeled dideoxynucleotides.

What is gene editing?

Experimentally altering a gene sequence within cloned DNA or living cells.

What does CRISPR-Cas technology do?

It allows for the editing of genes in living cells by using a guide RNA to direct the Cas9 enzyme.

What happens after Cas9 cuts the DNA?

Two repair events are possible: nonhomologous end joining (NHEJ) or homologous recombination repair (HRR).

What is the result of repair by nonhomologous end joining (NHEJ)?

It may incur a small deletion that inactivates the gene.

What is needed for homologous recombination repair (HRR)?

Donor DNA homologous to the target region carrying the desired mutation.

What is the significance of PCR in genetics?

PCR allows for the amplification of specific DNA segments from complex mixtures.

How does RT-PCR differ from traditional PCR?

RT-PCR amplifies RNA by first converting it into cDNA using reverse transcriptase.

What is the role of primers in PCR?

Primers bind to the DNA strands to initiate the synthesis of new DNA strands.

What is the purpose of using dideoxynucleotides in DNA sequencing?

They terminate DNA strand growth, allowing for the determination of the sequence.

What is the function of the fluorescence detector in Real-Time PCR?

It measures the changes in fluorescence emitted during the PCR reaction.

What does the term 'Cycle Threshold (Ct)' refer to?

The point at which the fluorescence signal is detectable above the background noise.

What is the typical outcome of PCR after 30 cycles?

A target DNA sequence will increase 230-fold (~1 billion-fold).

What is the advantage of using CRISPR-Cas technology?

It provides a precise method for editing genes in living organisms.

What is the significance of amplifying chromosomal DNA nonspecifically?

It allows for the amplification of most chromosomal DNA from very small samples.

What is the role of the spacer region in sgRNA?

It is complementary to the target gene and guides Cas9 to the correct location for editing.