Recombinant DNA and DNA Purification Techniques

These flashcards cover key concepts and definitions related to recombinant DNA and DNA purification techniques discussed in the lecture.

Asilomar Conference (1975)

A conference that led to specific recommendations for recombinant DNA research due to concerns about unknown risks.

Recombinant DNA

DNA that has been artificially created by combining DNA from different organisms.

Biosafety guidelines for rDNA research

Recommendations to ensure safe practices during recombinant DNA research, including the establishment of oversight committees.

Ethanol and salt in DNA purification

Used in protocols to precipitate DNA by neutralizing charges and lowering the dielectric constant.

Phenol extraction

A method of removing proteins from DNA solution based on phase separation, where DNA remains in the aqueous phase.

RNA vs DNA isolation

RNA isolation is more difficult due to RNA's instability and sensitivity to RNases, requiring special handling.

Spectrophotometry

A method for determining DNA concentration by measuring UV absorbance at 260 nm.

Agarose gel electrophoresis

A technique that separates DNA fragments by size, providing information on fragment size, integrity, and verification of PCR products.

Silica matrix purification

A method where DNA binds to silica in high salt conditions, allowing for the removal of contaminants.

Ancient DNA (aDNA)

DNA extracted from ancient biological materials, which faces challenges such as degradation and contamination.

DNA concentration conversion

The process of converting DNA concentration from ng/µL to molarity using molecular weight calculations.

What information can gel electrophoresis provide that spectrophotometry cannot?

Fragment size, DNA integrity, number of fragments, and verification of digestion of PRC products

Why is DNA length required to calculate molarity?

Molecular weight depends on the number of base pairs

What information is needed to convert DNA concentration from ng/uL to molarity?

DNA length, molecular weight, and volume

What does a low A260/230 ratio indicate?

Contamination from salts, phenol, or other organic compouds

What does a low A260/280 ratio (~1.5 suggest)

Protein contamination

What does an A260/28- ratio indicate?

Pure DNA

What DNA concentration corresponds to A260 = 1

~50 ug/ml for ds

How can gel electrophoresis estimate DNA concentration?

By comparing band intensity to known standards

Why are fluorescent DNA-binding dyes more accurate than spectrophotometry?

They specifically bind DNA, reducing interference from contaminants.

How does spectrophotometry measure DNA concentration?

By measuring absorbance at 260 nm, where DNA absorbs UV light.

What special precautions are needed for RNA isolation?

Use RNase-free reagents, sterile technique, and often include DNase treatment to remove DNA contamination.

Why is RNA more difficult to work with than DNA?

RNA is less stable due to its 2’ OH group and is highly susceptible to degradation by ubiquitous RNases.

How does phenol extraction separate proteins from DNA?

Phenol denatures proteins and partitions them into the organic phase, while DNA remains in the aqueous phase due to its polarity.

How is ancient DNA typically analyzed?

By extracting DNA from preserved samples, amplifying it with PCR, and sequencing it.

What are the main challenges in studying ancient DNA?

Degradation (fragmentation), contamination with modern DNA, chemical damage, and low quantity.

How does a silica column selectively bind DNA during purification?

In high-salt conditions, DNA binds to silica while contaminants are washed away; DNA is later eluted in low-salt conditions.

Why must chromosomal DNA be handled gently during purification?

Because it is very large and prone to shearing, which can fragment the DNA and compromise its integrity.

Why are salt and ethanol used together to precipitate DNA?

Salt neutralizes the negative charges on DNA, and ethanol reduces solubility, allowing DNA to aggregate and precipitate out of solution.

Why does Kuzma argue that the process of creating GMOs matters, not just the final product?

Because different processes can introduce different unintended effects, risks, and ethical concerns that are not detectable by evaluating the final product alone.

Is recombinant DNA research allowed in the U.S., and under what conditions?

Yes; it is regulated under NIH guidelines requiring risk assessment, appropriate biosafety levels, and Institutional Biosafety Committee (IBC) approval.

List key recommendations from the Asilomar Conference.

• Biological containment (e.g., weakened host strains)

• Physical containment (biosafety levels)

• Restriction of high-risk experiments

• Institutional oversight (biosafety committees)

What were the main goals of the Asilomar Conference recommendations?

To ensure safe recombinant DNA research through biosafety guidelines, containment strategies, and oversight.

What scientific development led to the 1975 Asilomar Conference on recombinant DNA?

The discovery that DNA could be cut and recombined between organisms using restriction enzymes and ligase, enabling recombinant DNA and raising biosafety concerns.