Green with envy?? Jelly fish “GFP” Transformed vertebrates

DNA Manipulation, Gene Cloning and DNA Microarrays

Gene Cloning Techniques for gene cloning enable scientists to prepare multiple identical copies of gene- sized pieces of DNA. Most methods for cloning pieces of DNA share certain general features. –For example, a foreign gene is inserted into a bacterial plasmid and this recombinant DNA molecule is returned to a bacterial cell. –Every time this cell reproduces, the recombinant plasmid is replicated as well and passed on to its descendents. –Under suitable conditions, the bacterial clone will make the protein encoded by the foreign gene.

One goal may be to produce a protein product for use. A second goal may be to prepare many copies of the gene itself. –This may enable scientists to determine the gene’s nucleotide sequence or provide an organism with a new metabolic capability by transferring a gene from another organism.

Restriction Enzymes In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria. Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences.

Different restriction enzymes cut DNA in different ways. –each enzyme has a different restriction site

Restriction enzyme cleaves DNA at specific sequence of bases called a restriction site. –often a symmetrical series of four to eight bases on both strands running in opposite directions. –restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3 Restriction enzymes cut the sugar phosphate backbone bonds of both DNA strands, creating single-stranded ends, sticky ends. –These extensions will form hydrogen-bonded base pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme

Recombinant plasmids--produced by splicing restriction fragments from foreign DNA into plasmids. –Plasmid is a circular piece of DNA found in bacteria and contain genes. –Plasmids can be used to insert DNA from another organism into a bacterial cell. Then, as a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it. Recombinant DNA vectors Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Cloning a gene into a bacterial plasmid can be divided into five steps. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig Blue colonies White colonies

When the source of DNA is small or impure, the polymerase chain reaction (PCR) is quicker and more selective. (limitation of PCR -- produces short DNA segments within a gene and not entire genes.) This technique can quickly amplify any piece of DNA without using cells. Devised in 1985, PCR has had a major impact on biological research and technology. The polymerase chain reaction (PCR) clones DNA entirely in vitro Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

The DNA is incubated in a test tube with special DNA polymerase, a supply of nucleotides, and short pieces of single- stranded DNA as a primer. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 20.7

PCR can make billions of copies of a targeted DNA segment in a few hours. –This is faster than cloning via recombinant bacteria. PCR, a three-step cycle: –heating, –cooling, –replication, –brings about a chain reaction that produces an exponential number of DNA molecules. PCR is so specific and powerful that only minute amounts of DNA need be present in the starting material

PCR

PCR has amplified DNA from a variety of sources: –fragments of ancient DNA from a 40,000-year- old frozen wooly mammoth, –DNA from tiny amount of blood or semen found at the scenes of violent crimes, –DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders, –DNA of viral genes from cells infected with difficult-to-detect viruses such as HIV. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

DNA Microarrays

Basic Steps to Microarray: Obtain cells with genes that are needed for analysis. Isolate the mRNA using extraction buffer. –Remove the buffer with RNA. –The mRNA will be 5’-3’ with repeating AAA..end. Convert each mRNA into colored cDNA. –Targets labeled with fluorophores.Targetsfluorophores

Basic Steps to Microarray: With mRNA degraded reverse cDNA created.reverse Mix colored samples of cDNA.

Microarray or DNA Chip: Every spot on the chip represents a different coding sequence from different genes. Each spot on the chip is made of a DNA probe that can pair with the cDNA that was created. probe

Basic Steps to Microarray: Incubation with the mixed cDNA and the chip DNA will yield some pairing. Wash off unbound cDNA to see what has bound to the microarray.

Visualizing Bound cDNA: The slide with the microarray chip is placed inside a dark box where it is scanned with a high resolution laser that detects the bound fluorescent labels. –The information and images are then sent to the computer for analysis.

Analyzing the Data: Creates a ratio image. Green images signal expression in one condition. Red images signal expression in one condition. Yellow images signal expression in both conditions.

Definitions: Target - the nucleic acid (cDNA) sample who’s identity and quantity are being measured.Target Fluorophore – usually green and red labels attached to the target to enable visualizing expression.Fluorophore Microarray works as reverse hybridization method converting from mRNA to cDNA 3’-5’ with TTTT…end.Microarray Probe – an attached nucleic acid with a known sequence (the DNA chip).Probe