DNA Analysis and Manipulation Flashcards

Flashcards covering key concepts and techniques in analyzing and manipulating DNA and RNA.

Recombinant DNA

The ability to manipulate DNA with precision in a test tube or organism.

Manipulation 1

Cleavage of DNA at specific sites by restriction nucleases.

Manipulation 2

DNA ligation, which joins DNA molecules from different sources.

Manipulation 3

DNA cloning (through cloning vectors or PCR).

Manipulation 4

Nucleic acid hybridization, identifying DNA/RNA sequences.

Manipulation 5

Chemically synthesizing DNA molecules with any sequence.

Manipulation 6

Rapid determination of the sequence of nucleotides in DNA or RNA.

Gel Electrophoresis

Separates DNA molecules of different sizes.

Negatively charged DNA

Move towards the positive charged electrode in Gel Electrophoresis.

Larger DNA fragments

Migrate more slowly in gel electrophoresis.

Agarose

Polysaccharide isolated from seaweed, used for DNA > 500 nucleotide pairs.

Polyacrylamide gels

Used for DNA fragments < 500 nucleotides long.

Pulsed-field gel electrophoresis

Separates extremely long DNA molecules; changes the electric field periodically.

Changing electric field in pulsed-field gel electrophoresis

Reorient molecules before continuing to move through the gel.

Ethidium bromide

Stains DNA in gels, fluoresces under UV light.

DNA cloning

Making many identical copies of a DNA molecule.

DNA cloning accomplishment

Inserting a DNA fragment into a self-replicating genetic element.

Self-replicating genetic element

Usually a plasmid.

Purified plasmid DNA circles

First cut with a restriction nuclease to create linear molecules.

DNA ligase

Enzyme used to covalently join the DNA to be cloned to the plasmid.

Recombinant DNA circle

Introduced back into bacterial cells.

Bacterial cells

Grow and divide, replicating recombinant plasmids.

Result of process on page 10

Double-stranded cDNA copy of original mRNA.

Denaturation

Losing function due to unfolding structure.

Hydrogen bonds

Breaks during denaturation, but no covalent bonds.

Denaturation as a result of heating

Melting.

Hyperchromic shift

Increase in UV absorption of heated DNA in solution.

Melting temperature (Tm)

Point at which 50% of strands are unwound/denatured.

Denaturation

Heating DNA to around 90°C.

Lowering temperature

Complementary strands re-form a double helix.

Hybridization

DNA renaturation.

PCR (invented in the 1980s)

Revolutionized DNA/RNA analysis.

PCR function

Allows DNA from a specified region to be greatly amplified.

Primer

Provides a 3' end from which synthesis can begin.

PCR primers

Synthesized chemically, hybridize to genomic DNA.

Newly synthesized DNA molecules

Serve as templates for the next round of replication.

Cell-free cloning

Automation of the whole procedure.

Process on page 17

Blood sample from infected person -> extract RNA -> Reverse transcription and PCR amplification of HIV cDNA -> Gel Electrophoresis.

STRs analyzed in DNA

Short tandem repeats (e.g., CACACA… or GTGTGT…).

Where STRs are found

Positions (loci) in the human genome.

Number of repeats in each STR locus

Ranges from 4 to 40 in different individuals.

Variability in STR sequences

Individuals will usually inherit a different number of repeats at each STR locus from their mother and from their father.

Sanger sequencing

Dideoxy sequencing.

Deep RNA sequencing (RNA-seq)

Sequencing the entire repertoire of RNA from a cell or tissue.

Genome annotation

Attempts to mark out genes and ascribe a role to each.

Nucleic acids absorb ultraviolet (UV) light

Most strongly at wavelengths of 254 to 260 nm.

Peak absorption by DNA & RNA

Occurs at 260 nm.

UV light

Used in the localization, isolation, and characterization of molecules that contain nitrogenous bases.

Sedimentation Velocity Centrifugation

Uses ultraviolet absorption optics to monitor the migration of molecules during centrifugation and determine the velocity of sedimentation.

Fluorescence in situ Hybridization (FISH)

Combined with fluorescent probes to monitor hybridization.