Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Chapter 13 An Introduction to Cloning and Recombinant DNA

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Cloned Libraries A collection of cloned DNA sequences from one source Excellent resource Specific genes can be recovered using a labeled nucleic acid probe Fig

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Genomic libraries Collection of clones containing (in theory) at least one copy of every gene in genome. Choice of vector and host Includes all DNA - coding and non-coding sequences Collection of ALL DNA in the genome

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning cDNA libraries Snapshot of all mRNAs present in cell/tissue at given time. Extract RNA, reverse transcribe, make cDNA, Insert into appropriate vector Screen for presence of clone Representation of all mRNAs present in cell at given time

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Differences between genomic and cDNA libraries genomic cDNA libraries from all cells have same content libraries from different cells have different content genes represented relatively equally genes represented depending on expression levels regulatory sequences present - promoter, introns no regulatory sequences present all genes representednot all genes represented

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Using Probes Bacterial colonies, each derived from a single cell, grow on culture plate Each colony about 1mm across Nitrocellulose or nylon filter is placed on the plate Some cells of each colony adhere to it; the filter mirrors colony distribution on plate Fig a,b

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Fig d Filter lifted off; put into a solution Cells stuck to it rupture; cellular DNA sticks to the filter; DNA denatured to single strands at each site Radioactively labeled probe added to the filter; probe binds to DNA fragments with a complementary base sequence Using Probes, continued

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Fig e Locate probe by exposing filter to x-ray film Image on the film reveals location of the colony that has the gene of interest Using Probes, continued

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Example of “positives”

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning How to get many copies of DNA from very small amounts Polymerase chain reaction or PCR Amplify specific sequence of interest from large pool

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Kary Mullis1993Nobel Prize

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning PCR Fig a-c

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning PCR, continued Fig d,e

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Fig f,g PCR, continued

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Billions of Fragments Are Rapidly Synthesized through PCR Fig h

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Exponential amplification after 1 round: 1 copy to 2 copies after 2 rounds: 2 copies to 4 copies after 30 cycles: 2 30 = ~1 billion copies

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning

Geothermal pool in Lower Geyser basin, Yellowstone Nat’l Park Thermus aquaticus Growth range is 50-80ºC ( ºF); optimum is 70ºC (158ºF)

Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning PCR machines