Chapter 20—DNA Technology &...

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?


3. Introduction of the cloning vector into cells (transformation)


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)

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.


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.


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.


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


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?


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)?

Chapter 20—DNA Technology &...

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