AMPLIFYING AND ANALYZING DNA

Amplifying DNA a. Recombinant DNA

AMPLIFYING DNA B. Polymerase Chain Reaction (PCR) You would clone the gene using the polymerase chain reaction. The steps would include a. selecting two primers that would flank the gene b. heating the DNA to separate the strands c. reducing the temperature and annealing the primers to the DNA strands d. raising the temperature and extending the primers using Taq polymerase e. repeating this cycle a number of times

GEL ELECTROPHORESIS Gel electrophoresis is the movement of charged particles under the influence of an electric field. It is used to separate macromolecules like nucleic acids and proteins on the basis of size and charge (like a sieve) DNA is a negatively charged particle…why? DNA fragments are made using restriction enzymes cutting the DNA strand at specific locations. Depending on where they cut, they form different length segments that migrate up to certain points on across the gel

EXAMPLE USE TTCC RESTRICTION ENZYME DNA SAMPLE ONE: ACTGTTCCACACTTCCGCACATCTTAATTCC

METHODS FOR ESTABLISHING PATTERNS IN DNA A. USING NON-CODING DNA EXAMPLE ONE INDIVIDUAL: TTAAGGTTAAGGTTAAGGTTAAGGTTAAGG TTAAGGTTAAGG

Sanger Method The Sanger sequencing reaction. Single stranded DNA is amplified in the presence of fluorescently labelled ddNTPs that serve to terminate the reaction and label all the fragments of DNA produced. The fragments of DNA are then separated via polyacrylamide gel electrophoresis and the sequence read using a laser beam and computer. a primer of known sequence is required for each sequencing reaction. Thus, one cannot take any piece of DNA and “just sequence it.” A known starting point, and thus some knowledge of the sequence, is required to begin the reaction.

C. SANGER TECHNIQUE Dideoxynucleotides (dd-A, dd-T, dd-C, dd-G) lack a 3’ OH group on the deoxyribose therefore if it is added to the growing DNA segment, then another nucleotide can not be attached to that segment and therefore terminating the segment prematurely. Fragments of the DNA segment are made varying in length. Gel electrophoresis is used to separate the fragments according to size. The bands indicate where a certain nucleotide is located on the segment of DNA. To analyze the entire sequence, you can run parallel gels using dd-A, dd-T, dd-C, and dd-G.

TEMPLATE T C G C A T A G C C

STRAND A G C G T A T C G G A G C T

Newer Modification An electropherogram of a finished sequencing reaction. As the fragments from the sequencing reaction are resolved via electrophoresis, a laser reads the fluorescence of each fragment (blue, green, red or yellow) and compiles the data into an image. Each colour, or fluorescence intensity, represents a different nucleotide (e.g. blue for C) and reveals where that nucleotide is in the sequence.

A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. 1) DNA (genomic or other source) is digested with a restriction enzyme and separated by gel electrophoresis, usually an agarose gel. Because there are so many different restriction fragments on the gel, it usually appears as a smear rather than discrete bands. The DNA is denature into single strands by incubation with NaOH. 2) The DNA is transfered to a membrane which is a sheet of special blotting paper. The DNA fragementsw retain the same pattern of separation they had on the gel. 3) The blot is incubated with many copies of a probe which is single-stranded DNA. This probe will form base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it (e.g. alkaline phosphatase or horseradish peroxidase). 4) The location of the probe is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen or gives off light which will expose X-ray film. If the probe was labeled with radioactivity, it can expose X-ray film directly.