chapter 20—dna technology & genomics€¦ · • dna fingerprinting. genomic...
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Chapter 20—DNA Technology
& Genomics
If we can, should we?
Biotechnology• Genetic manipulation
of organisms or their
components to make
useful products
– Humans have been doing this for 1,000s of
years
– plant & animal
breeding
Biotechnology Today
• Genetic Engineering
– Direct manipulation of DNA for practical purposes
– Why do this?
• diagnose diseases or defects
• cure diseases or defects
• change/improve organisms
Gene Cloning
Why clone genes? 1. to produce a protein product
2. to prepare many copies of the gene itself
Recombinant DNA
• Contains genes from 2 different sources– Made with restriction
enzymes • Enzymes made by
bacteria that cut DNA at specific locations (why?)
• hundreds of different enzymes
– (EcoRI, HindIII, BamHI, SmaI)
• cuts at a restriction site– specific sequence of
DNA– symmetrical
“palindrome”
– produces sticky ends
Genes can be cloned in vectors called plasmids� Plasmids
� small, self-replicating circular DNA molecules� naturally occur in bacteria� insert DNA sequence (gene of interest) into plasmid
� Transformation
� insert recombinant plasmid into bacteria� culture (grow) recombinant bacteria = cell clones � permits production of multiple copies of a specific gene or DNA
sequence (gene clones)
Why use bacteria?
Cloning a Human Gene
using a bacterial
plasmid
5 Steps:
1. Isolation of vector and gene-source DNA
2. Insertion of DNA into the vector
Other possibilities?
Cloning a Human Gene
3. Introduction of the cloning vector into cells (transformation)
Cloning a Human Gene
4. Cloning of cells (and foreign genes)
Use both ampicillin resistance & color to identify clones containing recombinant plasmids:
� only clones containing plasmids will survive ampicillin antibiotic on plate
� only clones with disrupted lacZ
gene (& foreign DNA) will be white (functional gene turns X-gal blue)
5. Identification of cell clones carrying the gene of interest (next slide ☺☺☺☺)
Using a nucleic acid probe to identify a cloned “gene of interest”
Probe� short, single stranded DNA molecule� mix with denatured DNA (single stranded)
DNA Hybridization� probe bonds to complementary DNA
sequence (gene of interest)Label� probe is labeled for easy detection
(radioactivity or fluorescence)
But bacteria do it differently…
Eukaryotic gene…In a bacterial cell…
see any problems?
� non-recognizable euk. promoter
� no RNA processing
Solutions:
� Expression vectors (use prokaryotic promoters)
� cDNA (gene lacking introns)
� mature mRNA → DNA
(using reverse transcriptase)
� Yeast Artificial Chromosomes (YACs)
Essay Qs
• DNA Sequencing
• DNA to Protein (Transcription & Translation)
• Cloning a Gene
• DNA Fingerprinting
Genomic Libraries—store cloned genes
Can store an entire genome in a genomic
library
(1,000s of cloned
genes)
Polymerase Chain Reaction—Clones
DNA in vitro
Any piece of DNA can be copied many times WITHOUT using cells
Need:
� Sequence of DNA to be copied
� DNA primers (crucial)
� Heat-resistant DNA polymerase
� Nucleotides (G, A, C, T)
II. DNA Analysis & Genomics
• So we’ve got the DNA, now what?
Gel Electrophoresis—� works on nucleic acids (DNA/RNA) and proteins
� separates fragments by size, electrical charge,
etc. (different rates of movement through a gel in an electric field)
restriction fragment patterns
distinguish between DNA
Differences in DNA sequences = different cuts made by restriction enzymes = different bands on the electrophoresis gel
Can detect subtle differences between DNA sequences/alleles
Restriction Fragment Length
Polymorphisms (RFLPs)
• Differences in non-coding
DNA sequences that can
result in different patterns
of restriction fragment
lengths
• Detected/analyzed with
Southern blotting
• Useful as genetic markers
for making linkage maps
Southern Blotting (restriction fragment analysis)
Used to compare the DNA of different individuals or species
Can look at differences in specific genes (using DNA probes)
Human Genome Project
• Three stages:
– Genetic (Linkage) Mapping
– Physical Mapping
– DNA Sequencing• 3.2 billion base pairs
DNA Sequencing (Sanger method)
DNA Sequencing (Sanger method)
DNA Sequencing (Sanger method)
DNA Sequencing (Sanger method)
Strategies for Genome Sequencing
Chromosome Walking
Physical Mapping
Essay Qs
• DNA Sequencing
• DNA to Protein (Transcription & Translation)
• Cloning a Gene
• DNA Fingerprinting
Studying Gene Expression
DNA Microarrays (DNA Chips)
tell us…
Which genes are “active” and when
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
1. The principal problem with inserting an unmodified mammalian gene into the bacterial chromosome, and then
getting that gene expressed, is that
a) prokaryotes use a different genetic code from that of
eukaryotes.
b) bacteria translate polycistronic messages only.
c) bacteria cannot remove eukaryotic introns.
d) bacterial RNA polymerase cannot make RNA complementary
to mammalian DNA.
e) bacterial DNA is not found in a membrane-enclosed nucleus
and is therefore incompatible with mammalian DNA.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
2. Which of the following statements is consistent with the results below? *
a) B is the child of
A and C.
b) C is the child of
A and B.
c) D is the child of
B and C.
d) A is the child of
B and C.
e) A is the child of
C and D.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
3. Which of the following statements is most likely true?
a) D is the child of
A and C.
b) D is the child of
A and B.
c) D is the child of
B and C.
d) A is the child of
C and D.
e) B is the child of
A and C.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
4. Which of the following are probably siblings?
a) A and B
b) A and C
c) A and D
d) C and D
e) B and D
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
5. The segment of DNA shown in the figure below has restriction sites I and II, which create
restriction fragments A, B, and C. Which of the gels produced
by electrophoresis shown below would represent the separation
and identity of these fragments?
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
6. This restriction fragment contains a gene whose recessive allele is lethal. The normal allele has restriction sites for the restriction enzyme PSTI at sites I and II. The recessive allele lacks restriction site I. An individual who had a sister with the lethal trait is being tested to determine if he is a carrier of that allele. Indicate which of these band patterns would be produced on a gel if he is a carrier (heterozygous for the gene)?