Chapter 20 Techniques of Molecular Biology

The methods of molecular biology depend upon and were developed from an understanding of the properties of biological macromolecules themselves.

Part I NUCLEIC ACID

NUCLEIC ACIDS DNA and RNA separation by gel electrophoresis Principle: Linear DNA molecules migrate through the gel toward the positive pole with different rates when subject to an electrical field. The DNA molecules can be visualized by staining the gel with fluorescent dyes, such as ethidium.

NUCLEIC ACIDS Two matrices: polyacrylamide and agarose. Plyacrylamide has more resoving power. Pulsed-field gel electrophoresis for long DNAs (up to several Mb in length).

NUCLEIC ACIDS According to RNA it is similar, however RNA sample should be treated with reagents,e.g. glyoxal to prevent the formation of base pairs.

NUCLEIC ACIDS Restriction Endonuleases Cleaves DNA Molecules at Particular Sites Restriction enzymes recognize short target sequences and cut at a defined position within those sequences. They can generate different ends: flush ends and staggered ends. We use them to break large DNA into manageable fragments.

NUCLEIC ACIDS Recognition sequences and cut sites of various endonucleases

How we name them?? Take EcoRI for example: Eco: E. coli I: the first one

NUCLEIC ACIDS Hybridization probes can identify electrophoretically separated DNA and RNA Southern blot named after Edward Southern: DNA fragments, generated by digestion of a DNA molecule by a restriction enzyme, are run out on an agarose gel. Once stained, a pattern of fragments is seen. When transferred to a filter and probed with a DNA fragment homologous to just one sequence in the digested molecule, a single band is seen, corresponding to the position on the gel of the fragment containing that sequence.

NUCLEIC ACIDS One example of southern blot

NUCLEIC ACIDS DNA Cloning Some terms: DNA cloning; vector; insert DNA; library: a population of identical vectors that each contains a different DNA insert.

NUCLEIC ACIDS Characteristics of vector DNAs: 1.an origin of replication 2.a selectable marker 3.sigle sites for one or more restriction enzymes.

NUCLEIC ACIDS How to clone DNA in plasmid vectors: A fragment of DNA, generated by cleavage with a certain restriction enzyme, is inserted into the plasmid vector linearized by the same enzyme. The recombinant plasmid is introduced int o bacteria by transformation. Cells containing the plasmid can be selected by growth on the antibiotic to which the plasmid confers resistance.

NUCLEIC ACIDS Construction of a genomic DNA library: Genomic DNA and vector DNA, digested with the same restriction enzyme, are incubated together with ligase The resulting pool or library of hybrid vectors is then introduced into E. coli, and the cells are plated onto a filter placed over agar medium. The filter is removed from the plate and prepared for hybridization.

NUCLEIC ACIDS

Construction of a cDNA library Isolate mRNA use reverse transcriptase to synthesize complementary DNA strand from mRNA, then use DNA Pol I to synthesize double stranded DNA. Clone these cDNAs into appropriate vector (usually plasmid or phage) Use Oligo dT primer to hybridize to polyA tail of mRNA. Primer used by reverse transcriptase for extension. Reverse transcriptase is a DNA polymerase which uses RNA as a template to synthesize complementary DNA. Cloned from RNA viruses.

NUCLEIC ACIDS We should note that: No introns cloned, nor regulatory sequences Genes cloned in this method are only those that were expressed in the particular tissue mRNA was isolated from.

NUCLEIC ACIDS

After having constructed a DNA library, whether genomic or cDNA, we can use probes to find specific clones we are interested in.

NUCLEIC ACIDS Site-directed mutagenesis Using site-directed mutagenesis the information in the genetic material can be changed. A synthetic DNA fragment is used as a tool for changing one particular code word in the DNA molecule. This reprogrammed DNA molecule can direct the synthesis of a protein with an exchanged amino acid.

NUCLEIC ACIDS Polymerase Chain Reaction The Royal Swedish Academy of Sciences awards 1993’s Nobel Prize in Chemistry to: For more, click

NUCLEIC ACIDS for contributions to the developments of methods within DNA-based chemistry for his invention of the polymerase chain reaction (PCR) method for his fundamental contributions to the establishment of oligonucleotide-based, site- directed mutagenesis and its development for protein studies

Denaturation at 94 ℃ : the double strand melts open to single stranded DNA, all enzymatic reactions stop. Annealing at 54 ℃ : The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Extension at 72 ℃ : This is the ideal working temperature for the polymerase. The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template) NUCLEIC ACIDS Let’s look into it in more details:

NUCLEIC ACIDS

How to determine the sequence of bases in a DNA molecule The most commonly used method of sequencing DNA - the dideoxy or chain termination method - was developed by Fred Sanger in 1977 (for which he won his second Nobel Prize). The key to the method is the use of modified bases called dideoxy bases; when a piece of DNA is being replicated and a dideoxy base is incorporated into the new chain, it stops the replication reaction.

NUCLEIC ACIDS The Nobel Prize in Chemistry 1980 For more, click

NUCLEIC ACIDS Elements: The DNA to be sequenced: in single- stranded form; as a template. The four nucleotides The enzyme DNA polymerase and a primer A nucleotide analogue that cannot be extended and thus acts as a chain terminator

NUCLEIC ACIDS Dideoxynucleotides used in DNA sequencing

NUCLEIC ACIDS Train termination in the presence of dideoxynucleotides

NUCLEIC ACIDS Mechanism:

NUCLEIC ACIDS

One example of fluorecent chain-terminating nucleotides:

NUCLEIC ACIDS Sequencing Whole Genomes

NUCLEIC ACIDS First, the source clone is fragmented, producing a random mixture, and a random sub-clone is selected for sequencing by the Sanger method. To ensure that that the whole source clone has been sequenced, this stretch of DNA must be sequenced numerous times to produce an ordered overlapping sequence. Gaps in this process will occur where a sub- clone is not fully sequenced.

NUCLEIC ACIDS Contigs: Assemble the short sequences from random shotgun DNAs into larger contiguous sequences.

NUCLEIC ACIDS Contigs are linked by sequencing the ends of large DNA fragments

NUCLEIC ACIDS Genome-wide analyses Animal genomes contain complex exon-intron structure, so it is more difficult to find protein coding genes.

NUCLEIC ACIDS A variety of bioinformatics tools are required to identify genes and determine the genetic composition of complex genomes. A notable limitation of current gene finder programs is the failure to identify promoters EST (expressed sequence tag) is simply a short sequence read from a larger cDNA.

NUCLEIC ACIDS Gene finder methods: Analysis of protein–coding regions in Ciona

NUCLEIC ACIDS Comparative Genome Analysis Permits a direct assessment of changes in gene structure and sequence arisen during evolution. Refines the identification of protein-coding genes within a given genome.

NUCLEIC ACIDS What we have learned from comparative genome analysis Synteny: conservation in genetic linkage, between distantly related animals.

Part II PROTEINS

Purification of proteins To purify proteins we make use of their inherent similarities and differences. Protein similarity is used to purify them away from the other non-protein contaminants. Differences are used to purify one protein from another. Proteins vary from each other in size, shape, charge, hydrophobicity, solubility, and biological activity. PROTEINS

ImmunoAffinity Chromatography PROTEINS

Affinity Chromatography column matrix has a ligand that specifically binds a protein specialty affinity columns for binding recombinant proteins with certain "tags"

Affinity Chromatography PROTEINS

Ion Exchange Chromatography proteins have charges due to amino acid side groups bind to charged column matrix depending on their charge at a particular pH anionic--negatively charged: phosphocellulose, heparin sepharose, S-sepharose cationic--positively charged: DEAE-sepharose, Q- sepharose elute bound proteins from column based on charge and displacement by salt or pH

Ion Exchange Chromatography PROTEINS

Gel filtration Chromatography PROTEINS

Separation of proteins on polyacrylamide gels PROTEINS

Proteins to be isolated should be treated with sodium dodecyl sulphate (SDS) and a reducing agent first to eliminate the secondary, tertiary, and quarternary structure. PROTEINS

Protein molecules can be directly sequenced. Edman degradation Tandem mass spectrometry PROTEINS

PITC is used to derivitize the free N-terminus trifluoroacetic acid causes cleavage of the N- terminal amino acid from the protein acid treatment rearranges derivitized aa to stable PTH amino acid the PTH amino acid is separated by chromatography (HPLC) and identified N-terminus may be subjected to another round of degradation Edman degradation

PROTEINS

Tandem mass spectrometry

PROTEINS Proteomics Proteomics is the large-scale study of proteins, particularly their structures and functions. This term was coined to make an analogy with genomics. The availability of whole genome sequences in combination with analytic methods for protein separation and identification has ushered in the field of proteomics.

PROTEINS Proteomics is based on three principal methods: 2-D gel electrophoresis for protein separation Mass spectrometry for the precise determination of the molecular weight and identity of a protein Bioinformatics for assigning proteins and peptides to the predicted products of protein coding sequences in the genome.