molecular methods

MOLECULAR METHODS FOR

MOLECULAR GENETICS

MISBAH AKRAM

Molecular biology techniques are common methods used in  molecular biology, biochemistry, genetics, and biophysics which generally involve manipulation and analysis of DNA , RNA, Protein, and Lipid.

CATEGORIES

• Diagnostic• Analysis, Acquisition,Sequence• Variartions• Microarray methods• Proteomics

DIAGNOSTIC Basics of recombinant DNA technology Gel Electrophoresis Hybridization Sequencing PCR Chromosomal walking Karyotyping Immunoprecipitation Immunofluorescense Knockout / Homologous recombinantion Liposomes

BASICS OF RECOMBINANT DNA TECHNOLOGY

Why is it necessary to clone genes?

• Naturally occurring DNA molecules are very long and a single molecule usually carrying many genes

• Genes may occupy only a small proportion of the chromosomal DNA and the rest are noncoding sequence (a human gene might constitute only 1/1,000,000 of a chromosomal DNA)

Making cDNA from an

Eukaryotic Gene

• To express an eukaryotic gene into its product in bacterial cells, the mRNA is converted into cDNA

• The cloned cDNA can be expressed into protein product by inserting it into a plasmid containing a functional promoter (expression vector)

Purposes of Cloning Genomic DNA and cDNA

• Genomic DNA: Studying structures of genes, gene families Identifying promoters and other regulatory elements Studying evolution of genes

• cDNA: Studying structures of mRNA Measuring the levels of mRNA Studying developmental stage-specific and tissue-specific

expression of genes Studying processing and stability of mRNA Producing recombinant proteins

• Detection of Nucleic Acids

Agarose gel electrophoresis Hybridization, Southern blot and RNA

northern blot analyses

Agarose Gel Electrophoresis to Separate DNA

• Agarose is an inert carbohydrate isolated from seaweeds. DNA molecules of different sizes can be separated in an agarose gel by electrophoresis and visualized by staining the gel with ethedium bromide & observe under UV light. The DNA fragment can be recovered from the gel by extraction with phenol and chloroform

DNA Fragments Visualized Under UV Light

DNA on agarose gel is stained with ethedium bromide and observed under UV light

Hybridization• Hybridization: Two fragments of single-stranded

homologous DNA molecules can form hybrid through hydrogen-bonding formation (base pairing)

5’-GTACTTAGGCAATTGGGCA-3’ 3’-CATGAATCCGTTAACCCGT-5’

• If one of these two strands of DNA is labeled with radioactive isotopes, the hybrid will be easily visualized by autoradiography• Hybridization can occur between two homologous DNA

molecules or a DNA molecule and a RNA molecule• Southern blot and RNA northern blot hybridization

SOUTHERN BLOT

NORTHERN BLOT

WESTERN BLOT

Fluoerescence in situ Hybridization

This technique is used for cytological localization of molecules in the cell

Colony Hybridization• This method is

based on the principle that two homologous strands of nucleic acids can form hybrid form

• If one of the strands of nucleic acid is radio-labeled, the hybrid can be visualized by autoradiography

A Single Strand DNA to be Sequenced

Assigned Reading: Nobel lecture by F. Sanger on “DNA sequencing”, 1980

Separating the Products on a Denaturing Polyacrylamid Gel

3’

POLYMERASE CHAIN REACTION

CHROMOSOMAL WALKING

Chromosome Walking

• This technique allows the isolation of a long eukaryotic gene

• An alternative is to construct a BAC library that contains long piece DNA molecules

chromosome walking was used to identify a candidate gene for a disease like cystic fibrosis.

Nuclear Run-on Transcription Assay• Nuclear run-on assay can be

used to ascertain which gene is active in a given cell allowing transcription to continue in isolated nuclei

• Specific transcript can be identified by their hybridization to known DNAs on dot blot

• It can also be used to determine the effects of assay conditions on nuclear transcription

• Transcription activity of a specific gene can be determined

• It can also be used to measure template activity

Nuclear Run-On Assay to Measure the Transcription Rates of Genes in Various Tissues

MICROARRAY

• technique that allows you to determine the expression of many genes at one time

Principle of DNA Microarray

• Genes (cDNA or oligonucleotides are spotted on glass plates

• Messenger RNA is reverse transcribed into cDNA and labelled with Cy3 (emission 570nm, green) or Cy5 (emission 670 nm, red)

• Hybridization (mixing Cy3-cDNA and Cy5 cDNA)

• Scan the slide to detect the hybridization signals

APPLICATIONS

• Gene discovery: Examples Profiling of cancer-specific expressing genes Tissue-specific expression of genes Developmental-specific expression of genes

• Disease diagnosis: Collections of genes showing expression of genes specific

to certain types of diseases Examples: Specific cancer type, hematopoietic disease etc.

• Drug discovery: Pharmacogenomics To find correlations between therapeutic responses to drug

and gene profiles of patents• Toxicological research: Toxicogenomics• To find correlations between toxic responses to toxicants

and chages in the gene profiles of the objects exposed to such toxicants

Enlarged Photo of a Microarray Chip

• This array has 2400 human genes

• Red indicates increase of expression; yellow equal expression and green reduce of expression

• This technique can help to determine the profiles of gene expression

RNA-Seq Technology

RNA-seq, also called "Whole Transcriptome Shotgun Sequencing" ("WTSS"), refers to the use of high-

throughput technologies to sequence cDNA or RNA in order to get information about a sample's RNA content. The technique has been adopted in studies of diseases

like cancers. With deep coverage and base-level resolution, this technology provides information on

differential expression of genes, including gene alleles and differently spliced transcripts; non-coding RNAs;

post-transcriptional mutations or editing; and gene fusions

RNA-Seq Technology

Inactivation of a Gene by Using RNAi

• In vitro or in vivo production of siRNA

• Inhibition of mex3 mRNA by injecting siRNA into the cells

IMMUNOPRECIPITATION

• IP is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.

• This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.

• Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure.

KARYOTYPING

• Is the study of the structure and properties of chromosomes, chromosomal behaviour during mitosis and meiosis, chromosomal influence on the phenotype and the factors that cause chromosomal changes.

Nomenclature of chromosomes

PREPARATION OF CHROMOSOMES

KARYOTYPE PREPARATION

• Cultured cells are arrested at metaphase by adding colchicine

• This is when cells are more condensed and easiest to identify

• Arrested cells are broken up

• Metaphase chromosomes are fixed and stained.

• Chromosomes are photographed through microscopePhotograph of chromosome is cut up and arranged to form karyotype diagram.

HUMAN KARYOTYPE

POSITIONAL CLONING

• Technique that is used in genetic screening to identify specific areas of interest in the genome , and then determine what they do.

• Genes related to conditions such as Huntington's Disease and cystic fibrosis have been identified with this technique.

Positional Cloning- identification the cystic fibrosis (CF) gene:

Most common lethal genetic disease in the U.S. (~1 in 2,000).

First human gene identified by positional cloning.

Required 4 years and the work of many laboratories.

OVERVIEW OF CYSTIC FIBROSIS:

CF results from defect in protein that regulates the movement of salt and water in and out of cells.

Causes thick mucus secretions in the lungs, pancreas, and intestines.

Causes lung disease and organ failure, patients experience chronic bacterial infections.

Life expectancy is abut 40 years.

LIPOSOMES

• Liposomes are spherical, self-closed structures formed by one or several concentric lipid bilayers with an aqueous phase inside and between the lipid bilayers.

• Their ability to entrap different water-soluble compounds within the inner aqueous phase and lipophilic agents between liposomal bilayers has made them useful for delivery of different kinds of drugs.

• Liposomal modification with (PEG) increases their field of usage by enhancing circulation time and attachment of antibodies or different targeting moieties to their surface to targe.

• considered for intravascular drug delivery, using cells and noncellular components as the targeted sites for diagnosing and treating the most important cardiac pathologies, including myocardial infarction, coronary thrombosis, and atherosclerosis  at specific affected areas.

RANDOM PRIMING TECHNIQUE

DNA is denatured into single strands annealed to random hexamer oligonucleotides. These random primers can then be extended using

Klenow enzyme Random pieces of DNA for can be used for probe

production. These probes can be used on blots or DNA

microarrays.