Chapter 14 The Techniques of Molecular Genetics © John Wiley & Sons, Inc

Chapter 14The Techniques of Molecular

Genetics

© John Wiley & Sons, Inc.

Chapter Outline

Basic Techniques used to Identify, Amplify, and Clone Genes

The Molecular Analysis of DNA, RNA, and Protein


Basic Techniques Used to Identify, Amplify, and Clone

GenesRecombinant DNA, gene

cloning, and DNA amplification techniques allow scientists to

isolate and characterize essentially any DNA sequence

from any organism.


Gene Cloning

Gene cloning is the isolation and amplification of a given gene.

A recombinant DNA molecule is a DNA molecule made by joining two or more different DNA molecules (Gene and plasmid).


Amplification of a Gene In Vivo

A minichromosome carrying the gene of interest is produced in the test tube.

The recombinant minichromosome is introduced into a host cell (such as E. coli), and the host cell replicates the minichromosome.


Amplification of a Gene In Vitro

Short DNA strands complementary to DNA sequences on either side of the gene of interest are synthesized.

These short DNA strands are used to initiate the amplification of the gene by a heat-stable DNA polymerase in the polymerase chain reaction (PCR).


Restriction Endonucleases

Restriction endonucleases make site-specific cuts in DNA.

The nucleotide sequences are called restriction sites.

Restriction endonucleases protect bacteria from foreign DNA.

Bacteria protect endogenous restriction sites by methylation.

Restriction enzymes commonly recognize palindromic sequences. (it is equal to its reverse complement)


http://en.wikipedia.org/wiki/Reverse_complement



Structure of an EcoRI-DNA Complex


Many Restriction Endonucleases Make Staggered (Sticky)Cuts


When DNA is cleaved with a restriction endonuclease that makes “sticky” cuts, all of the resulting restriction fragments have complementary single-stranded termini.

The complementary single-stranded termini can hydrogen bond with each other and be joined together by DNA ligase.


Construction of Recombinant DNA Molecules In Vitro





“Vector" is an agent/system that can carry a DNA fragment into a host cell.

If it is used for reproducing the DNA fragment, it is called a "cloning vector". If it is used for expressing certain gene in the DNA fragment, it is called an "expression vector".

Plasmid (Vectors)

Plasmid (Vectors) Circular, double-stranded circular DNA

molecules present in bacteria. Range from 1 kb to over 200 kb. Replicate autonomously. Many carry antibiotic-resistance genes, which

can be used as selectable markers. Many useful cloning vectors were derived

from plasmid pBR322.


Bacteriophage (Phage lambda)

Vectors Most bacteriophage cloning vectors have

been constructed from the phage chromosome.

The central one-third (about 15 kb) of the chromosome contains genes required for lysogeny but not for lytic growth (lysogenic pase-prophage-lysogen).

This portion of the chromosome can be excised and replaced with foreign DNA.

The foreign DNA inserted must be 10-15 kb.


Phagemid Vectors

Contain components from phage chromosomes and plasmids (pBluescript II)

Replicate in E. coli as double-stranded plasmids.

Addition of a helper phage causes the phagemid to switch to the phage mode of replication, resulting in the packaging of single-stranded DNA into phage heads.


pBluescript II:--hybrid (phage and plasmids)--produce a vector that can grow as -----a plasmid-----produce a single stranded DNA in viral particles--unique restriction sites

http://en.wikipedia.org/wiki/Filamentous_phage

http://en.wikipedia.org/wiki/Plasmid


The Blue-White Color Test

The E. coli lacZ gene encodes -galactosidase.

-galactosidase converts the colorless substrate Xgal into a blue product.

Cells with -galactosidase activity produce blue colonies when grown on Xgal; cells lacking -galactosidase activity produce white colonies.


Shuttle (Eukaryotic) Vectors Because different organisms use different origins

of replication and regulatory signals, different cloning vectors must be used in different species.

Special cloning vectors can replicate in other prokaryotes and in eukaryotes.

Shuttle vectors can replicate in E. coli and in another species and it can propagate in two different host species


Importance/significance

Insulin

Antibodies

Growth Factors

Enzymes

The Polymerase Chain Reaction (PCR)

Synthetic nucleotides complementary to known flanking sequences “fragment of nucleotides” are used to prime enzymatic amplification of the sequence of interest “small fragment of nucleotides.”

Three repeated steps– Denaturation of genomic DNA (92-95°C)– Annealing of denatured DNA to oligonucleotide primers (50-60°C)– Replication (Elongation) of the DNA segment between the sites

complementary to the primers (70-72°C)

Amplification occurs exponentially; each cycle doubles the number of molecules of the sequence of interest.


Taq Polymerase DNA polymerase from Thermus aquaticus is used for

PCR because it is heat-stable.

Taq polymerase lacks proofreading activity, so errors are introduced into the amplified DNA at low but significant frequencies.– When high fidelity is required, heat-stable polymerases with

proofreading activity are used (Pfu or Tli).

Taq is amplifies fragments of DNA larger than a few thousand base pairs inefficiently.– For amplification of long segments of DNA (up to 35 kb), Tfl

polymerase is used.


Applications of PCR

Diagnosis of inherited human diseases (e.g., prenatal diagnosis).

Identification of individuals in forensic cases from small DNA samples.



1kb~1 min

nucleotides



Synthesis of Double-Stranded cDNAs from mRNA (also RT-PCR)


Amplification of Recombinant DNA

Antibiotic-sensitive recipient cells are transformed with the recombinant DNA molecule.

Transformed cells are selected by growth under conditions requiring the presence of a selectable marker present on the recombinant DNA molecule (usually an antibiotic).

The recombinant DNA molecule is amplified by the host cell ( e.g., insulin)


Techniques Necessary for Sequencing DNA

Restriction enzymes to prepare homogenous samples of specific segments of chromosomes.

Gel electrophoresis procedures able to resolve DNA fragments differing in length by a single nucleotide.

Gene-cloning techniques allowing preparation of large quantities of a DNA molecule.

Sanger sequencing Technique is used to determine nucleotide sequences.


DNA SequencingA population of DNA fragments is

generated.– One end is common to all fragments (the 5’

end of the sequencing primer).

– The other end terminates at all possible positions (the 3’ terminus).


2',3'-Dideoxyribonucleoside Triphosphates


Automated DNA Sequencing Fluorescent dyes (Cy5, Cy3) are used for detection of

DNA chains instead of radioactive isotopes (32P ,35S).

Products of all four chain terminator reactions are separated through a single gel or capillary tube.

Photocells detect fluorescence of the dyes as they pass through the gel or capillary tube.

Output of the photocell is directly transferred to a computer for analysis.


Automated Sanger DNA Sequencing






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Chapter 14 The Techniques of Molecular Genetics © John Wiley & Sons, Inc