Laboratory Techniques in DNA Manipulation: Lecture 1

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Questions and Answers

Oligonucleotide primers are short, single-stranded DNA fragments that are ______ to a region at one of the extremities of the DNA you want to amplify.

complementary

Oligonucleotide primers can bind to both single-stranded and double-stranded DNA.

False

What is the primary function of oligonucleotide primers in PCR?

To bind to the polymerase enzyme

To cut the DNA at specific locations

To provide a starting point for DNA polymerase to synthesize new DNA (correct)

To separate the DNA strands

Why is it important for oligonucleotide primers to be specific to the target DNA sequence?

Specificity ensures that the primers only bind to the desired DNA sequence and prevent amplification of unintended regions. Signup and view all the answers

Match the following primer design considerations with their descriptions:

Specificity of Sequence = The primer sequence should match the target DNA sequence. Absence of Dimerization Capability = Primers should not bind to themselves or each other. Absence of Significant Hairpin Formation = Primers should avoid forming loop structures within themselves. Avoid Secondary Priming Sites = Primers should not bind to unintended locations in the DNA template. Signup and view all the answers

Long oligonucleotide primers are generally more specific than short primers.

True Signup and view all the answers

The ______ is the temperature at which 50% of the primer is bound to the target DNA.

melting temperature (Tm) Signup and view all the answers

What are the major challenges associated with designing oligonucleotide primers for PCR?

Challenges include ensuring primer specificity, avoiding self-dimerization and hairpin formation, and preventing secondary priming sites. Signup and view all the answers

What is the primary function of Taq polymerase in PCR?

To synthesize new DNA strands Signup and view all the answers

PCR technology utilizes high temperatures that can denature most enzymes, but Taq polymerase can withstand these temperatures.

True Signup and view all the answers

What are the three key steps involved in the PCR process?

Denaturation, annealing, and extension Signup and view all the answers

The optimal temperature for the extension phase of PCR is ____°C.

72 Signup and view all the answers

What is the purpose of oligonucleotide primers in PCR?

To bind to the target DNA Signup and view all the answers

Thermocycling in PCR involves repeating a series of temperature changes approximately 10 times.

False Signup and view all the answers

Name one method used to confirm the success of the PCR reaction.

Gel electrophoresis Signup and view all the answers

Match the elements of the PCR reaction with their roles:

DNA template = The sequence to be amplified DNA polymerase = Enzyme that catalyzes DNA synthesis Nucleotides = Building blocks of DNA Buffer = Maintains optimal conditions for the enzyme Signup and view all the answers

What is the purpose of inserting the GST gene into pBluescript?

To allow bacteria to produce GST protein Signup and view all the answers

Restriction enzymes are used to insert genes into plasmids.

False Signup and view all the answers

Name one type of restriction enzyme mentioned in the content.

BamHI Signup and view all the answers

The GST gene is inserted into the __________ in pBluescript.

Multiple Cloning Site (MCS) Signup and view all the answers

Match the restriction enzymes with their recognition sequences:

BamHI = GGATCC EcoRI = GAATTC HindIII = AAGCTT NotI = GCGGCCGC Signup and view all the answers

What is the first step in engineering pBluescript to contain the GST gene?

Cut open the pBluescript DNA Signup and view all the answers

What role does DNA ligase play in the engineering of pBluescript?

DNA ligase sticks the DNA together after insertion. Signup and view all the answers

The multiple cloning site (MCS) is located within the β-galactosidase gene of pBluescript.

True Signup and view all the answers

The annealing temperature (Ta) is typically set ______ °C below the Tm of the primer.

5 Signup and view all the answers

Increasing the GC content of a primer will decrease its melting temperature (Tm).

False Signup and view all the answers

Which of the following factors can influence the Tm of a primer?

Length of the primer Signup and view all the answers

What is the primary function of a GC clamp in a primer?

A GC clamp, typically a guanine or cytosine base at the 3' end of the primer, improves binding stability at the target site. Signup and view all the answers

Match the following terms to their correct definitions:

Tm = The temperature at which 50% of the primer is bound to the target DNA Annealing temperature (Ta) = The temperature used during the annealing step of PCR GC clamp = A guanine or cytosine base at the 3' end of a primer to improve binding stability Signup and view all the answers

If the annealing temperature (Ta) is too high, what is the likely result?

Primers may not bind at all, leading to no amplification Signup and view all the answers

Oligonucleotides are ______ artificially based on the desired sequence.

synthesized Signup and view all the answers

Explain why the ability to customize oligonucleotide sequences is important for molecular cloning.

Customizing oligonucleotide sequences allows us to prepare oligonucleotides for specific cloning purposes. We can make mutations, add pieces of DNA to the ends, or design primers with specific features to facilitate cloning procedures. Signup and view all the answers

What is the primary role of DNA ligase in molecular biology?

To facilitate the bonding of DNA strands Signup and view all the answers

Blunt ends are more likely to join two DNA strands together than sticky ends.

False Signup and view all the answers

What are the names of the two non-compatible restriction enzymes mentioned?

HindIII and BamHI Signup and view all the answers

DNA ligase catalyzes the formation of a __________ bond.

phosphodiester Signup and view all the answers

Why do we generally use sticky end restriction enzymes in cloning?

Because they produce compatible sequences Signup and view all the answers

Match the following processes or concepts with their descriptions:

Ligation = Joining DNA strands through phosphodiester bonds Sticky ends = DNA ends with a single-stranded overhang Restriction enzymes = Enzymes that cut DNA at specific sequences PCR product preparation = Engineering DNA fragments for compatibility Signup and view all the answers

What must be engineered into the PCR product to ensure it can ligate with the pBluescript?

BamHI and HindIII restriction sites Signup and view all the answers

Sticky ends can easily be joined together without any additional modifications.

True Signup and view all the answers

What is defined as the number of atoms in 1/12th g of C12?

Mole Signup and view all the answers

1 mol of hydrogen is equal to 2 grams.

False Signup and view all the answers

What is the unit of measurement for amount of substance in moles?

mol Signup and view all the answers

The __________ is the amount of substance dissolved in a fluid.

concentration Signup and view all the answers

Which of the following represents the relationship between moles and molarity?

M = mol/L Signup and view all the answers

Match the following substances with their respective molecular weights:

Hydrogen (H) = 1 g/mol Carbon (C) = 12 g/mol Sodium Chloride (NaCl) = 58.44 g/mol Tris = 121.1 g/mol Signup and view all the answers

1 M of NaCl solution is equivalent to __________ g per liter.

58.44 Signup and view all the answers

To prepare a 100 mL solution, you should always start from solid form of the solute.

False Signup and view all the answers

Study Notes