Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Chapter 13An Introduction to Cloning and
Recombinant DNA
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Cloned Libraries
• A collection ofcloned DNAsequences fromone source
• Excellent resource• Specific genes can
be recoveredusing a labelednucleic acid probe
Fig. 13.14
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Genomic libraries
Collection of clones containing (in theory) at least one copyof 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 cellshave same content
libraries from different cellshave different content
genes represented relativelyequally
genes represented depending onexpression levels
regulatory sequences present - promoter, introns
no regulatory sequences present
all genes represented not all genes represented
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Using Probes
• Bacterial colonies,each derived from asingle cell, grow onculture plate
• Each colony about1mm across
• Nitrocellulose or nylon filter is placed onthe plate
• Some cells of each colony adhere to it; thefilter mirrors colony distribution on plate
Fig. 13.15a,b
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Fig. 13.15d
• Filter lifted off; put into a solution• Cells stuck to it rupture; cellular DNA sticks
to the filter; DNA denatured to singlestrands at each site
• Radioactively labeled probe added to thefilter; probe binds to DNA fragments with acomplementary base sequence
Using Probes,continued
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Fig. 13.15e
• Locate probe by exposing filterto x-ray film
• Image on the film revealslocation of the colony that hasthe 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 verysmall 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 Mullis 1993 Nobel Prize
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
PCR
Fig. 13.16a-c
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
PCR, continued
Fig. 13.16d,e
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Fig. 13.16f,g
PCR, continued
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Billions of Fragments Are RapidlySynthesized through PCR
Fig. 13.16h
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: 230 = ~1 billion copies
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
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 (122-176ºF); optimum is 70ºC (158ºF)
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
PCR machines