Transcript Chapter 1 A Perspective on Human Genetics

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. 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 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
cDNA
genomic
libraries from all cells
have same content
libraries from different cells
have different content
all genes represented
not all genes represented
regulatory sequences
present - promoter, introns
no regulatory sequences present
genes represented relatively
equally
genes represented depending on
expression levels
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
Fig. 13.15a,b
• Nitrocellulose or nylon filter is placed on
the plate
• Some cells of each colony adhere to it; the
filter mirrors colony distribution on plate
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Using Probes,
continued
Fig. 13.15d
• 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
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Using Probes, continued
• Locate probe by exposing filter
to x-ray film
• Image on the film reveals
location of the colony that has
the gene of interest
Fig. 13.15e
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 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
PCR, continued
Fig. 13.16f,g
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Billions of Fragments Are Rapidly
Synthesized 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
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning