Exploring Genes

EXPLORING GENES Chapter 6

Basic Tools in Gene Exploration • Restriction Enzymes- molecular scissors • Blotting techniques-The Southern and Northern Blots • DNA sequencing • Solid phase synthesis of nucleic acids • The polymerase chain reaction (RT-PCR)

Restriction Enzymes Split DNA into Specific Fragments • also called restriction endonucleases • recognize specific base sequences in double-helical DNA and cleave at specific places, both strands of DNA containing the recognized sequences • most recognize specific sequences of four to eight base pairs and hydrolyze a phosphodiester bond in each strands • mostly recognize palindromes or inverted repeats

Restriction enzyme from Streptomyces achromogenes

Restriction enzymes are used to cleave DNA molecules into specific fragments that are more readily analyzed and manipulated than the entire parent molecule

Restriction fragments can be separated by gel electropresis and visualized • The electrophoretic mobility of a DNA is inversely proportional to the number of base pairs. • Polyacrylamide gels are used to separate fragments containing about as many as 1000 base pairs • Agarose gels are used to separate mixtures of bigger fragments • Bands can be visualized by autoradiography or staining with ethidium bromide

Southern Blotting

DNA is usually sequenced by controlled termination of replication (Sanger Dideoxy Method) • Sanger Dideoxy Methods of DNA synthesis generates fragments based on the last base in the sequence • Based on selective interruption of DNA synthesis

DNA Probes and Genes Can Be Synthesized by Automated SolidPhase Methods • Synthesis of DNA strands is thru the sequential addition of an ACTIVATED monomer to a growing chain that is linked to a solid support • The activated monomers are the protonated deoxyribonucleoside3’-phosphoramidites

Uses • Synthesized oligonucleotide labeled at end with 32P or a flourescent tag can be used to search for a complementary sequence in a very long DNA molecule or even in a genome consisting of many chromosome • Synthesis of new tailor made genes

Selected DNA Sequences Can Be Greatly Amplified by the Polymerase Chain Reaction (PCR) PCR cycle consists of three steps 1. Strands separation 2.Hybridization of the primers 3. DNA synthesis

PCR is a powerful technique in medical diagnostics, forensics and molecular evolution • PCR can detect bacteria and virus with the use of primers • PCR is a promising method for the early detection of cancer • PCR is use in forensic and legal medicine because an individual DNA is highly distinctive • PCR can amplify DNA that remain intact for thousands of year so that ancient DNA can identified and characterized

Recombinant DNA Technology • Unrelated genes are combined in the laboratory • The resulting recombinant DNA can be clone by introducing them into a suitable cells, where they are replicated by the DNA-synthesizing machinery of the host

Plasmids and Lambda Phage Are Choice Vectors for DNA Cloning in Bacteria • Plasmids are circular duplex DNA molecules occurring naturally in some bacteria

pBR322 • One of the most useful plasmids, which contains genes for tetracycline and ampicillin resistance • Contains restrictions sites for EcoR1, Sal1 and Pst1

Lambda (λ) Phage • Another widely used vector which can destroy its host or can become part of it

M13 Phage • Another useful vector for cloning DNA • Useful for sequencing the inserted DNA • Does not kill its bacterial host

Specific genes can be cloned from digests of genomic DNA

Long stretches of DNA can be efficiently analyzed by chromosome walking • Larger pieces of DNA can inserted into bacterial artificial chromosomes (BACS) or yeast aritifical chromosomes (YACS) • To scan long regions of DNA, overlaps in the library fragments are produced.

Manipulating the eukaryotic genes • Eukaryotic genes can be introduced into bacteria and the bacteria can be used as factories to produce a desired protein. • Introduction into animals provides the ability to examine gene action for gene therapy. • In regards to plants, inserted genes may make the plant pest resistant or enables to grow in harsh condition.

Complementary DNA prepared from mRNA can be expressed in host cells

Gene expression levels can be comprehensively examined

 New genes inserted into eukaryotic cells can be efficiently expressed

Recombinant DNA molecules can be introduced into animal cells by: 1. DNA molecules precipitated by calcium phosphate are taken up by animal cells 2. DNA is microinjected into a cell 3. Viruses are used to bring new genes into animal cells

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