Lab 7: pGLO DNA and Restriction DIgest

Genotype

determined by the DNA content passed to the organism by its parents

Genomics

study of genes

Genome Similarities

individuals of the same species usually have similarities in their genomes, but not all their genomic DNA sequences are identical because individuals have many genetic differences (except twins or individuals cloned from the same parent)

Bacterial Reproduction/Multiplication

parent DNA is replicated using parent DNA strand as template and the clones contain a direct copy of the DNA of the original parent bacterial cell

Bacterial Colony Observed on Agar Plate

consists of millions of bacteria cloned from single parent bacteria

Imperfect Replication

replication is not always perfect e.g. single nucleotide mutations in DNA

Phenotype

determined by observed physical and behavioral characteristics of the organism such as appearance, metabolic activity, and movement

Factors that Affect Phenotype: Appearance Examples

size, shape, color

Factors that Affect Phenotype: Metabolic Activity Examples

metabolize starch but not cellulose, able to degrade ampiciliin

Factors that Affect Phenotype: Movement Examples

walks or slides

Transcribed DNA Molecules

carry information that specifies the protein structure that build cells

Genome Expressed as Phenotype

allowed through mRNA translation to protein, protein-protein interactions, and cofactor availability

Mutations

a change in the nucleotide sequence of a short region of a genome that results in genome changes

Polymorphisms

mutations that do not cause disease and are present in >1% of the population, naturally occurring genetic differences among organisms in the same species

Most Common Type of Polymorphism

variation at a single DNA base pair

Silent Mutations

mutations that do not show a change in the translated amino acid and have no effect on phenotype

Nucleic Acid Quantification

performed before performing downstream experiments using the extracted DNA and RNA, based on Beer Lambert’s Law

DNA and RNA: Nucleic Acid Quantification

colorless and have a lambda max of 260 nm (in UV range)

Double Stranded DNA Average Extinction Coefficient

0.020 μg/mL^-1cm^-1 or 0.02 cm^-1 x ng^-1 x μL

Single Stranded RNA Average Extinction Coefficient

0.025 μg/mL^-1cm^-1

Why ng/μL is Used

very small quantities of DNA are used in molecular biology experiments and nucleotide sizes of the DNA fragments vary

Nucleic Acid Purity

can be tested by measuring absorbance at 260 and 280 nm, yields an estimate not a precise result, results can be affected by small pH changes in the solution and wavelength accuracy

Purity Indication for DNA: 260/280 Ratio, 1.8 - 2

pure DNA

Purity Indication for DNA: 260/280 Ratio, <1.8

presence of proteins, alcohol, acidic pH, or other contaminants

Purity Indication for DNA: 260/280 Ratio, >2

RNA contamination, basic pH

Purity Indication for DNA: 260/320 Ratio, 2.0 - 2.2

pure DNA

Purity Indication for DNA: 260/320 Ratio, <2

EDTA, carbohydrates and phenol

Restriction Enzymes

cut the DNA at specific palindromic sequence of base pairs, named according to the bacterial strain where the enzyme was first isolated

Restriction Site

palindromic sequence of DNA

Palindromic DNA Sequence

a DNA or RNA segment that has the same sequence when read from 5’ to 3’ on one strand and 5’ to 3’ on the complementary strand

DNA Restriction Fragments

can be cloned into DNA vectors such as plasmids to prepare recombinant DNA molecules ued for protein synthesis and gene analysis

Restriction Digests

useful to identify mutations in DNA sequences

Codon

three nucleotide sequence of RNA that codes for a particular amino acid, degenerate

Codons: Degenerate

different codons can translate to the same amino acid

Mutagenesis

process by which a nucleotide changes in a DNA sequence

Lambda DNA

linear and double-stranded (duplex) DNA molecule that is isolated from bacteriophage, 48,502 base pairs in length, has Hind III restriction sites

Hind III Digest of Lambda DNA

used as a DNA marker to determine the size of unknown DNA fragments

DNA Shapes

DNA is very small and compact when coiled, when cut upon digestion with enzymes it unfolds and longer in size, DNA of most organisms is negatively supercoiled, DNA may become nicked during extraction and create hooks in circular DNA

Nucleic Acid Quantification: Beer Lambert’s Law

A is absorbance, c is the nucleic acid concentration in ng/μL, e is the wavelength-dependent extinction coefficient in (μg/mL)^-1cm^-1 and b is the pathlength in cm