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