Restriction Enzymes & Gel Electrophoresis

Genes and genomes

All the DNA in a cell = genome

Genomes vary in size and gene content

DNA encodes all the proteins necessary for the function of life

sequences of bases create genes

Genes code for amino acids

amino acids are the building blocks of proteins

DNA

Blank _____is contained in the nucleus of the cell

chromosomes are made of__________

somatic cells are diploid (2n)

2 chromosome sets

Gametic cells are haploid (n)\1 chromosome set

DNA Structure

Two strands of antiparallel polymers

Nucleotide bases

Adenine

Thymine

Guanine

Cytosine

5 carbon sugar (ribose)

Phosphate backbones

The strands are complementary

A pairs with T

C pairs with G

Plasmids

In addition to chromosomal DNA, most bacteria also have ____

Double stranded, circular DNA

smaller than the chromosome (just a few genes)

Typically code for a specific function that is not essential for the life of the cell (e.g. antibiotic resistance)

___are easily transferred between bactereia

Genetically engineered ___ are commercially available for experimental use

____ are commonly use as vectors for genetic cloning

-small in size

-easy to manipulate

molecular biologists clone genes into ____ to study structure and function

-can lead to production of artificial enzymes and pharmaceuticals, GMO crops, etc.

Restriction Endonucleases

Bacterial enzymes that cut double stranded DNA at specific sites unique to tje enzyme

Naturally used to protect from viral invasion by bacteriophages

cutting into pieces make the DNA inactive

Restriction sites

generally a palindromic sequence (same forwards and backwards)

Make a staggered cut: Creates single -stranded “sticky ends”

Recombinant Molecules

by using the same restriction enzyme to cut plasmid DNA and a gene, unrelated poeces of DNA can be stitched into a recombinant molecule

-re-inserted into the cell'

-proliferation

Restriction pattern analysis

Restriction fragment length polymorphism

Generate fragments of different lengths that separate on a gel in a restriction pattern

Gel Electrophoresis

Extensively used technique for separating macromolecules

-uses electricity to move charged molecules through gel matrix

-can separate fragments of DNA based on mass, sizem shape, or charge

-molecules separate into bands that can be seen with dye and stain

Gel Electrophoresis

Gel material: agarose

Polysaccaride derived from seaweed

dissolved in buffer (Tris-Acetate-EDTA)

1% concentration

-pore size in gel can be manipulated for different purposes by adjusting the perventage of agarose

Ethidium Bromide

dye stains DNA and fluoresces orange under UV light

solidifies in a gel plae

comb creates loading wells

Gel Electrophoresis

Electrodes on either end of the boat run current through buffer

-DNA (negatively charged) runs through the pores in the gel tpward the positive electrode

-speed depends on size of fragments