Dr. Kathleen Hill Office Hours: Monday noon to 5pm Room 333 Western Science Centre Hill Research and Teaching Website Study Guide for 2581b: WebCT site 281b Dr. Hill

What is the function of soap, salt and alcohol in DNA extraction? 2581b Hill C9

In the laboratory a common method of DNA extraction uses SDS (soap) to soluabilize membranes and inactivate proteins and Proteinase K to digest proteins and then extractions with Phenol: Chloroform to physically remove proteins from DNA. Salt and alcohol permit precipitation of DNA 2581b Hill C9

Recombinant DNA Technology Key Concepts Complementary Two key properties of nucleic acids 5’ 3’ 5’ Antiparallel ACGT TGCA ACGT TGCA 2581b Hill C9

Sequence specific binding A key property of Protein:nucleic acid interactions Recombinant DNA Technology Key Concepts 2581b Hill C9

Restriction Endonucleases Bind to Specific Palindromic DNA Sequences Palindrome (Rotational symmetry) Cuts: through covalent bonds in the sugar phosphate backbone of DNA –Blunt/flush -Blunt ends –Staggered -Single stranded “sticky ends” 3’ overhang 5’ overhang 2581b Hill C9

Fig b Hill C9 Certain restriction endonucleases cleave the covalent bonds of the sugar phosphate backbone at sites in the palindromic sequence that create Blunt or flush ends

Fig. 9.2.b 2581b Hill C9 Certain restriction endonucleases cleave the covalent bonds of the sugar phosphate backbone at sites in the palindromic sequence that create 5’ sticky or cohesive ends Hydrogen bonds possible Hybridization possible with complementary sequence

Fig. 9.2.c 2581b Hill C9 Certain restriction endonucleases cleave the covalent bonds of the sugar phosphate backbone at sites in the palindromic sequence that create 3’ sticky or cohesive ends Hydrogen bonds possible Hybridization possible with complementary sequence

2581b Hill C9 Restriction Endonucleases derived from prokaryotes have the Genus and species name of their prokaryote origin numbered since there may be many of these enzymes in a single species

2581b Hill C9 Restriction Endonucleases are derived from prokaryotes Restriction Endonucleases cut double stranded DNA at specific palindromic sequences Bacteria use restriction enzymes to protect from invading viral nucleic acids Bacteria methylate their DNA to protect it from digestion by their own restriction enzymes

Fig b Hill C9 ***Note Above: The assumption was made that A, C, T, G occur with equal frequencies in the DNA target sequence*** Determining the frequency that a palindrome is present in a DNA sequence

Fig b Hill C9 ***Note: you may be asked to determine the average restriction fragment size in cases where there are nonequivalent frequencies of the four nucleotides***

Fig b Hill C9 Restriction Endonucleases with shorter recognition sequences tend to have more frequent cut sites and produce shorter fragments Frequent cutter Less frequent cutter

Fig. 9.3.a 2581b Hill C9 1.The average fragment length for a four base cutter is 256 bp 2.The average fragment length for a six base cutter is about 4kb

Restriction Endonuclease Digestion Cutting DNA Circular DNA with one cut = one fragment Linear DNA with one cut = two fragments EcoRI 2581b Hill C9

Fig. 9.4.a The concept of a complete vs. partial digest Complete Digest: All possible sites cut in all templates in the reaction Partial Digest: The reaction is incomplete and not all sites and templates are cut 2581b Hill C9

A linear DNA molecule has two target sites for restriction enzyme AvaI. What is the maximum number of DNA lengths that can be produced if the sample is only partly digested? A. 3 B. 4 C. 5 D. 6 E b Hill C9

A linear DNA molecule has two target sites for restriction enzyme AvaI. What is the maximum number of DNA lengths that can be produced if the sample is only partly digested? A. 3 B. 4 C. 5 D. 6 E b Hill C9

A linear DNA molecule has two target sites for restriction enzyme AvaI. What is the maximum number of DNA lengths that can be produced if the sample is only partly digested? Question variables to be aware of: Linear or circular Number of target sites Type of digest: partial or complete 2581b Hill C9

Restriction Fragment Analysis Gel electrophoresis separates DNA fragments primarily on the basis of size/length 2581b Hill C9 Restriction Enzyme digest of plasmid DNA

Fig. 9.5.a DNA is electrophoresed through a polymer Agarose vs. Acrylamide Gels Agarose: larger migration space for DNA Polyacrylamide: smaller migration space for DNA 2581b Hill C9

Fig. 9.5.a DNA is loaded (pipetted) into the wells of the gel Sucrose or glycerol provide density so the DNA sample sinks into the wells of the submerged gel A dye helps to see the sample fall into the well DNA is negatively charged and migrates at a rate relative to its size/length 2581b Hill C9

Fig. 9.5.b.2 Size markers assist in determining fragment length 2581b Hill C9 Ethidium Bromide is a dye that intercalates with DNA and fluoresces upon UV exposure + - DNA Migration Ethidium Bromide Migration Anatomy of a DNA Gel UV Light

Fig. 9.5.b.2 Cutting a complex DNA sample with a frequent cutter results in a smear Size markers assist in determining fragment length 2581b Hill C9 Genomic DNA after Digestion