Lab Techniques James Chappell & Cheuk Ka Tong
Contents Page • Restriction Enzymes • Gel Electrophoresis • Blotting techniques-Southern, Northern and Western • DNA sequencing • Polymerase Chain Reaction (PCR) • Recombinant DNA • Gene Cloning • References/ Recommended Reading
Restriction Enzymes • Restriction Nuclease- An enzyme that cleaves a molecule of DNA at any site where a specific short sequence of nucleotides occurs. 2key types • Endonuclease- Cleaves within the DNA molecule • Exonuclease- Cleaves at the ends of the DNA molecule
Endonucleases • 4 types - classified on subunit composition, cleavage position, sequence-specify and co-factor requirement. • Type II is the main one that is used in gene cloning. Two key terms • Recognition sites – Nucleotide sequence that is recognised. • Cleavage sites- Phosphodiester bond that is cleaved.
5’----GAATTC----3’ 3’----CTTAAG----5’ Endonucleases • Make break the phosphodiester bond of each of the stands of the double helix. 5’----G-3’ 5’AATTC----3’ 3’----CTTAA--5’ 3’G----5’
Endonucleases-Recognition site • 3 Considerations- • Sequence – Determine specificity • Length of sequence- Determines frequency • Palindrome- sequence that reads the same backwards and forwards. • Isoshizomers – Restriction enzymes that recognise the same recognition site
EndonucleasesApplications • Allow specific cutting and removal of genes from a complex molecule of DNA. • Complementary sticky ends (cohesive ends) allow joining of DNA molecules. "The work on restriction nucleases not only permits us easily to construct recombinant DNA Molecules and to analyze individual genes but also has led us into the new era of synthetic biology where not only existing genes are described and analyzed but also new gene arrangements can be constructed and evaluated" ‘Nobel prizes and restriction enzymes’ in GENE (1971)
Gel Electrophoresis • Electrophoresis - the migration of charged molecules in an electric field though a solution or solid support • Various types – defined by support used • Paper – amino acids, small peptides • Polyacrylamide – Proteins, small DNA/RNA (<500bp) • Agarose – DNA/RNA • Good preparative and analytical method
Gel Electrophoresis • Gel electrophoresis uses a cross-linked polymers (agarose) that contain various pores. • Pores allow molecular sieving, where molecules e.g. DNA, can be separated based upon there mobility through the gel.
DNA Gel Electrophoresis Mobility = Charge + Molecular Dimensions • Charge per nucleic acid is constant • This means separation is based upon length of the DNA molecules and this is how we can separate and identify DNA molecules.
DNA Gel Electrophoresis • Linear DNA has a linear relationship to distance migration. • If add molecular markers of known mass can calculate mass of our fragment by plotting a linear plot.
DNA Gel Electrophoresis • Other factors determining mobility- • Polymer concentration e.g. Agarose • Conformation of DNA • Electrophoresis
DNA Gel Electrophoresis • Detection • Dye e.g. ethidium bromide • Audioradiography 32P, • Blotting (see later) • Uses • Analytical- Can determine size of DNA fragment, • Preparative – Can identify a specific fragment based on size
Blotting Techniques • Blotting – Transfer of DNA, RNA or Proteins, typically from a electrophoresis gel to a membrane e.g. nitrocellulose. This membrane can then be subject to further techniques such as hybridization. • Hybridization – Process where two complementary single strands of nucleic acid (DNA or RNA) form a double helix.
Blotting Techniques • Using specific probes that are labelled specific sequences of DNA can be identified. • There are three main hybridization techniques which vary in the sample blotted and the probes used; • Northern Blot-Transfer of an RNA sample separated and identified using DNA or RNA probes. • Southern Blot-Transfer of an DNA sample separated and identified using DNA or RNA probes. • Western Blot- Transfer of an Protein sample separated and identified typically using an antibody.
Applications • The main use of this technique is to identity any changes in DNA sequencing or genes expressed, e.g. comparing genes expressed by a diseased cell to genes expressed by an healthy cell. • Other uses include- Testing for hereditary disease, Evolutionary history of species, Screening e.g.food supply • Applications to synthetic biology - identification of various parts in natural organisms, -?more?
DNA Sequencing • DNA Sequencing – Determining the order of nucleotides in a DNA molecule • Key technique as it can give us information about a DNA molecule, e.g. location and order of genes, restriction sites. • In addition, for recombinant DNA gives verification of gene cloning experiments. • 2 possible use’s for project – Identify sequence of new part, - Checking recombinant DNA. • Various techniques are available. • The key technique used today is Dideoxy method.
DNA Sequencing Deoxyribonucleotide acid This is essentially the monomer of DNA. Polymerization of nucleotides occurs by condensation reaction of a 5’ phosphate to a 3’ hydroxyl group Dideoxyribonucleotide acid There is no 3’hydroxyl group to allow polymerization.
Polymerase Chain Reaction (PCR) • A method for amplifying specific DNA sequences. • Components required: - Target sequence - A pair of primers - dNTPs (ATGC) - DNA polymerase
PCR • One PCR cycle involves three steps: - Strand separation (95ºC) - Hybridization of primers (54ºC) - DNA synthesis (72ºC) • After n cycles, the sequence is amplified 2n-fold.
Polymerase Chain Reaction (PCR) • Five noteworthy features of PCR: • The sequence of the target need not be known. • The target can be much larger than the primers (>10 kb). • Primers do not have to perfectly match flanking sequences. • Stringency can be controlled by temperature and salt (MgCl2). • PCR is very sensitive.
Recombinant DNA Technology • The construction of new combinations of unrelated genes. • These novel combinations can be cloned and amplified by introducing them into host cells.
Recombinant DNA Technology • A DNA fragment of interest is covalently joined to a DNA vector. - A vector can replicate autonomously in an appropriate host. - Plasmids and phage λ are common vectors for cloning in E.coli.
Recombinant DNA Technology • The DNA fragment of interest and the plasmid vector are both cut using the same restriction enzyme. • The single-stranded ends of the fragment are complementary to those of the cut plasmid. • The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase.
Gene Cloning • One of the most useful plasmids for cloning is pBR322. • pBR322 contains genes for resistance to tetracycline and ampicillin. • Different endonucleases can cleave this plasmid at a variety of unique sites.
Gene Cloning • Insertion of DNA at the EcoRI site does not alter either of the genes for antibiotic resistance. • However insertion at the SalI or PstI site causes insertional inactivation. Basis for selection of cells containing recombinant DNA.
References/Recommended Readings • Chapter 8 – Manipulating Proteins, DNA and RNA - Molecular Biology of the Cell (4th Edition), Alberts, Johnson, Lewis, Raff, Roberts, Walter, Garland Science 2. Chapter 6- Exploring Genes -Biochemistry (5th Edition), Berg, Tymoczko, Stryer, Freeman