Chapter 8: Recombinant DNA Technology

Principal Points Cloning is the making of many copies of a segment of DNA  Cloning vectors range from plasmids to artificial chromosomes to viruses  Genomic libraries are collections

Chapter 8: Recombinant

DNA Technology

Linnea Fletcher Ph.D.

BIOL 2316

Principal Points

 Cloning is the making of many copies of a segment of DNA

 Cloning vectors range from plasmids to artificial chromosomes to viruses

 Genomic libraries are collections of clones that contain at least one copy of every DNA sequence in an organism’s genome

 cDNA made from mRNA represent eukaryotic genes without

introns

 Identification of clones can be done using cDNA probes,

antibodies, complementation of mutations, and sequencing

 The PCR is a procedure for amplifying a specific segment of

DNA

DNA fragments separated by gel electrophoresis and visualized under UV light (EtBr intercalates between the bases; allowing for visualization of

 What are the 3 general steps used to clone DNA?

 Isolate DNA from an organism

 Cut the organismal DNA and the vector with restriction enzymes making recombinant DNA

 Introduce the recombinant DNA into a host

 Restriction Enzymes

 Recognize a specific site (generally a pallidromic sequence)

 Produce overhangs or straight cuts

 Naturally found in bacteria, they protect against viruses and foreign DNA

 Named for the organism they from which they are isolated

 The first letter is that of the genus and the 2nd and 3rd are from the species

Figure 8.1 Restriction

site in DNA, showing

symmetry of the

sequence around the

center point The

sequence is a

palindrome, reading

the same from left to

right (5’-to-3’) on the

top strand (GAATTC,

here) as it does from

right to left (5’-to-3’)

on the bottom strand

Shown is the

restriction site for

EcoRI.

How are restriction

enzymes named?

Figure 8.2 Examples of how restriction enzymes cleave DNA (a) SmaI results in blunt ends (b) BamHI results in 5’ overhanging (“sticky”) ends (c) PstI results in 3’

overhanging (“sticky”) ends

Figure 8.3 Cleavage of DNA by the restriction enzyme EcoRI EcoRI makes

staggered, symmetrical cuts in DNA, leaving “sticky” ends A DNA fragment with a

sticky end produced by EcoRI digestion can bind by complementary base pairing (anneal) to any other DNA fragment with a sticky end produced by EcoRI cleavage

The gaps can then be sealed by DNA ligase

 What types of nucleic acids are used as cloning vectors?

 What are some elements that are necessary for a plasmid

cloning vector and in what ways is the pUC19 vector especially useful for cloning DNA?

 Why must a cloning plasmid be recircularized before

transforming a host bacterium?

 Describe how insertional mutagenesis is used to distinguish

nonrecombinant plasmid from recombinant plasmid when

screening transformed hosts.

 What are some elements that are necessary for a shuttle

plasmid cloning vector?

 What are some elements that are necessary for an expression cloning vector, a YAC, and a BAC?

 Compare the size of DNA insert that can be carried by a plasmid,

a YAC, and a BAC.

Must have:

1) Ori

2) A dominant selectable

Marker

3) Cleavage sites for cloning

4) (high copy no.)

Figure 8.4 The plasmid cloning

vector pUC19 This plasmid has

an origin of replication (ori), an

ampR selectable marker, and a

polylinker located within part of

the β-galactosidase gene lacZ+

Figure 8.5 Insertion of a piece of DNA into the plasmid cloning vector pUC19 to produce a

recombinant DNA molecule The vector pUC19 contains several unique restriction enzyme sites localized in a polylinker that are convenient for constructing recombinant DNA molecules The insertion of a DNA fragment into the polylinker disrupts part of the β-galactosidase (lacZ+) gene,

leading to nonfunctional β -galactosidase in E coli The blue–white color selection test described

in the text can be used to select for vectors with or without inserts

Yeast Artificial Chromosome

Example of a yeast artificial chromosome (YAC) cloning vector A YAC vector

contains a yeast telomere (TEL) at each end, a yeast centromere sequence (CEN),

a yeast selectable marker for each arm (here, TRP1 and URA3), a sequence that allows autonomous replication in yeast (ARS), and restriction sites for cloning

 What types of DNA inserts are found in genomic

libraries, chromosomal libraries, and a cDNA

library?

inserts for a genomic library are especially large?

constructing a chromosomal library?

 What are some advantages of constructing a cDNA library?

library and what special enzymes are required to prepare cDNA?

advantages do they offer?

Figure 8.7 Use of partial digestion with a restriction enzyme to produce DNA fragments of appropriate size for constructing a genomic library.

cDNA Libraries are best for eukaryotes

Figure 8.8 The synthesis of double-stranded complementary DNA (cDNA) from a polyadenylated mRNA, using reverse transcriptase, RNase H, DNA polymerase I, and DNA ligase

Cloning of cDNA

using BamHI

linkers

recombinant clone with a target gene in a cDNA

library cloned into an expression vector? (Western Blot)

screening strategy? (protein product will stick to the membane)

(radioactivity is an inexpensive method!)

Screening for specific

cDNA plasmids in a cDNA

Library by using an antibody probe

The antibody binds to a specific

Site on a protein that is made via

The inserted foreign DNA This

Is a Western Blot since it uses antibody

To detect a protein

 Southern Blot: Detection of A Specific DNA Sequence Using a Probe

 Once some sequence information is available on a target gene, how can it be quickly screened for in a genomic library?

 What is replica plating, and how does it make library screens more efficient?

 What are some requirements for probing DNA with a

complementary DNA fragment and how are these requirements met

in screening a library with a probe?

 Describe how DNA probes can be labeled for detection of annealing

to a complementary sequence Which method(s) have the greatest sensitivity of detection?

 About how long must a synthetic probe be in order to discriminate a target sequence in a genomic library?

 What is a heterologous probe, and what limitations are there to the use of these probes in screening genomic libraries?

 Describe how GenBank is used to design a heterologous probe that matches a consensus sequence.

 What can be done to design a probe specific to a target gene when there is no clear consensus sequence in the target gene?

Designing Probes

organism, a homologous probe can be made If it is known in another organism, a heterologous probe can be made

labeled with radioactive tags or fluorescent tags.

Genosys makes custom oligos)

Labeling of DNA

Using DNA probes to screen plasmidgenomic libraries for specific DNAsequences

Figure 8.11 Using DNA probes to

screen plasmid genomic libraries for

specific DNA sequences

Figure 8.11 Using DNA probes to

screen plasmid genomic libraries for

specific DNA sequences

Describe how

complementation can be

used to screen an

expression library

What are some limits to the

use of this approach for

isolating a specific gene?

Figure 8.12 Example of

cloning a gene by

complementation of

mutations: cloning of the

yeast ARG1 gene

fragments to be separated by size in gel

Figure 8.14 An agarose gel electrophoresis apparatus (right) and power supply (left)

Restriction Mapping

determined, and what is a practical application of

such a map?

in its genomic background Explain how this is

done.

such an analysis?

plastic membrane before probing?

Applications of a Map

determined, and what is a practical application of

such a map?

in its genomic background Explain how this is

done.

such an analysis?

plastic membrane before probing?

fragment can insert

into the pUC19 vector

in two alternative

orientations By

cutting with AatII, the

site for which is

Blots continued

different from a Southern blot?

blot analysis?

in an automated DNA procedure?

reveal?

Can you read this sequence?

TRY IT

A

Read from the bottomup

Figure 8.19 Autoradiogram of a dideoxy

sequencing gel The letters over the lanes

(A, C, G, and T) correspond to the

particular dideoxy nucleotide used in the

sequencing reaction analyzed in a given

lane

Figure 8.20 Results of automated DNA sequence analysis using fluorescent dyes The procedure is described in the text The automated sequencer generates the curves shown in the figure from the fluorescing bands on the gel The colors are generated by the machine and indicate the four bases: A is green, G is black, C is blue, and T is red Where bands cannot be distinguished clearly, an N is listed

what occurs at each of these temperature?

 What is RT-PCR, and in what ways is it different from PCR reactions?

11 The diagram above (Figure 1) shows the products from a Sanger method of enzymatic extension sequencing of DNA What is the sequence of the TEMPLATE strand?

c 5’-AGCGGTC-3’

d 5’-CTGGCGA-3’