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Chapter 20 Lecture

Concepts of Genetics Tenth Edition

Recombinant DNA Technology

Chapter Contents

20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors

20.2 DNA Libraries Are Collections of Cloned Sequences 20.3 The Polymerase Chain Reaction Is a Powerful Technique for

Copying DNA 20.4 Molecular Techniques for Analyzing DNA 20.5 DNA Sequencing Is the Ultimate Way to Characterize DNA

Structure at the Molecular Level

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20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction

Enzymes and DNA Cloning Vectors

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Section 20.1

• Recombinant DNA refers to the joining of DNA molecules, usually from different biological sources, that are not found together in nature

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Section 20.1

• The basic procedure for producing recombinant DNA involves – generating specific DNA fragments using restriction

enzymes – joining these fragments with a vector – transferring the recombinant DNA molecule to a host

cell to produce many copies that can be recovered from the host cell

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Section 20.1

• The recovered copies of a recombinant DNA molecule are referred to as clones and can be used to study the structure and orientation of the DNA

• Recombinant DNA technology is used to isolate, replicate, and analyze genes

• A restriction enzyme binds to DNA at a specific recognition sequence (restriction site) and cleaves the DNA to produce restriction fragments

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Section 20.1

• Most recognition sequences exhibit a form of symmetry described as a palindrome, and restriction enzymes cut the DNA in a characteristic cleavage pattern – Mostly four to six nucleotides long – Some contain eight or more nucleotides

• DNA ligase joins restriction fragments covalently to produce intact DNA molecules

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Section 20.1 • Vectors are carrier DNA molecules that can replicate

cloned DNA fragments in a host cell

• Vectors must be able to replicate independently and should have several restriction enzyme sites to allow insertion of a DNA fragment

• Vectors should carry a selectable gene marker to distinguish host cells that have taken them up from those that have not

• A plasmid is an extrachromosomal double-stranded DNA molecule that replicates independently from the chromosomes within bacterial cells 10

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Section 20.1

• Plasmids used for DNA cloning usually have been engineered to contain – a number of convenient restriction sites – a marker gene to select for its presence in the host cell

• Plasmids are introduced by the process of transformation

• Transformation is achieved through

– using calcium ions and brief heat shock to pulse DNA into cells – Electroporation, which uses a brief but high-intensity pulse of

electricity to move DNA into bacterial cells

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Section 20.1

• Both the plasmid DNA and DNA to be cloned are cut with the same restriction enzyme

• DNA restriction fragments from the DNA to be cloned are added to the linearized vector in the presence of DNA ligase

• A recombinant DNA is produced, which is then introduced into bacterial host cells by transformation

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Section 20.1

• Recombinant DNA can be readily identified by using selectable marker genes

• Genes that provide resistance to antibiotics such as ampicillin and genes such as the lacZ genes are very effective selectable markers

• Blue-white selection is used to identify cells containing recombinant and nonrecombinant DNA

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Section 20.1

• Phage vectors were among the earliest vectors used in addition to plasmids

• The central third of lambda (λ) phage vectors can be replaced with foreign DNA without affecting the ability to infect cells and replicate

• Lambda vectors can carry up to 45 kb of cloned DNA

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Section 20.1

• Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are two other examples of vectors that can be used to clone large fragments of DNA

• BACs are generally very large but low copy number (one to two copies/bacterial cell) plasmids

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Section 20.1

• Expression vectors are engineered to express a gene of interest to produce large quantities of the encoded protein in a host cell

• Expression vectors are available for both prokaryotic and eukaryotic host cells

• Plant and animal cells can serve as hosts for recombinant DNA, in addition to bacteria and yeast

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Section 20.1

• Rhizobium radiobacter (formerly Agrobacterium tumefaciens) can be used to transform plant cells with T-DNA containing foreign DNA

• Rhizobium contains the Ti plasmid (tumor inducing) with genes that induce tumors

• Tumor-inducing genes can be removed from the vector so the recombinant vector does not produce tumors

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Section 20.1

• The vector when mixed with plant cells enters inside the cell, and the foreign DNA gets inserted into the plant genome

• The plant cells can be grown in tissue culture and eventually regenerate a mature plant carrying a foreign gene

• These cells are then grown by tissue culture to give a mature plant carrying a foreign gene

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Section 20.1

• While E. coli is used as a prokaryotic host cell when working with plasmids, yeast is widely used as a host for DNA cloning and expression of eukaryotic genes because – it can be grown easily and manipulated – its genetics have been studied intensively – its entire genome has been sequenced – it can posttranslationally modify eukaryotic proteins – it is considered to be safe

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Section 20.1

• A variety of different human cell types can be grown in culture and used to express genes and proteins

• These lines can be subjected to various approaches for gene or protein functional analysis, including drug testing for effectiveness at blocking or influencing a particular recombinant protein being expressed, especially if the cell lines are of a human disease condition such as cancer

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20.2 DNA Libraries Are Collections of Cloned Sequences

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Section 20.2

• DNA libraries represent a collection of cloned DNA samples derived from a single source that could be a particular tissue type, cell type, or single individual

• A genomic library contains at least one copy of all the sequences in the genome of interest

• Genomic libraries are constructed by cutting genomic DNA with a restriction enzyme and ligating the fragments into vectors, which are chosen depending on the size of the genome

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Section 20.2

• YACs were commonly used to accommodate large sizes of DNA necessary to span the 3 billion bp of DNA in the human genome

• Whole-genome shotgun cloning approaches and new sequencing methodologies (next-generation sequencing) are replacing traditional genomic libraries

• All DNA fragments in a genomic sample are sequenced without the need to insert DNA fragments into vectors and cloning them in host cells

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Section 20.2

• Complementary DNA (cDNA) libraries contains complementary DNA copies made from the mRNAs present in a cell population and represents the genes that are transcriptionally active at the time the cells were collected for mRNA isolation

• A cDNA library is prepared by – isolating mRNA from cells – synthesizing the complementary DNA using reverse transcriptase – cloning the cDNA molecules into a vector

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Section 20.2

• Reverse transcriptase PCR (RT-PCR) can be used to generate cDNA from mRNA by – first making a single-stranded cDNA copy of the mRNAs using

reverse transcriptase – then using PCR to copy the single-stranded DNA into double-

stranded DNA

• The cDNA libraries provide an instant catalog of all the genes active in a cell at a specific time and have been very valuable tools for scientists isolating and studying genes in a particular tissue

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Section 20.2

• Library screening is used to sort through a library and isolate specific genes of interest

• Probes are used to screen a library to recover clones of a specific gene. A probe is any DNA or RNA sequence that is complementary to the target gene of sequence to be identified

• To screen a plasmid library, clones from the library are grown on agar plates to produce colonies. The colonies are screened by transferring bacterial colonies from the plate to a filter and hybridizing the filter with a nucleic acid probe to the DNA sequence of interest

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Section 20.2

• The colony corresponding to the one the probe identified on the filter is identified and recovered

• A phage library is screened by plaque hybridization

• Libraries enable scientists to clone DNA and then identify individual genes in the library

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20.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA

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Section 20.3

• The polymerase chain reaction (PCR) copies a specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities

• PCR is a rapid method of DNA cloning that eliminates the need to use host cells for cloning

• The double-stranded DNA to be cloned is put in a tube with DNA polymerase, Mg2+, and the four dNTPs

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Section 20.3

• PCR requires two oligonucleotide primers (short, single-stranded sequences), one complementary to the 5' end of one strand of the target DNA and another complementary to the 3' end of the other strand

• The primers anneal to denatured DNA, and the complementary strands are synthesized by a heat-stable DNA polymerase

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Section 20.3

• The three steps of PCR—denaturation, primer annealing, and extension—are repeated over and over using a thermocycler to amplify the DNA exponentially

• The DNA strand is doubled in each cycle, and the new strands along with the old strand serve as templates in the next cycle

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Section 20.3

• A limitation of PCR is that some information about the nucleotide sequence of the target DNA must be known in order to synthesize the primer

• Minor contamination from other sources can cause problems

• PCR cannot amplify long segments of DNA

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Section 20.3

• PCR is a very useful tool since it allows the screening of mutations involved in in genetic disorders

• The location and nature of a mutation can be determined quickly

• Allele-specific probes for genetic testing can be synthesized; PCR is important for diagnosing genetic disorders

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Section 20.3

• PCR techniques are particularly advantageous when studying samples from single cells, fossils, or a crime scene, where a single hair or even a saliva-moistened postage stamp in the source of DNA

• PCR has been used to enforce the worldwide ban on the sale of certain whale products and determination of pedigree background of purebred dogs

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Section 20.3

• Reverse transcription PCR (RT-PCR) is used to study gene expression by studying mRNA production by cells or tissues

• Quantitative real-time PCR (qPCR) or real-time PCR allows researchers to quantify amplification reactions as they occur in ‘real time’ – The procedure uses an SYBR green dye and TaqMan probes,

which contain two dyes

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20.4 Molecular Techniques for Analyzing DNA

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Section 20.4

• A restriction map establishes the number and order of restriction sites and the distance between restriction sites on a cloned DNA segment

• It provides information about the length of the cloned insert and the location of restriction sites within the clone

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Section 20.4

• Restriction maps were created by cutting DNA with different restriction enzymes and separating the DNA fragments by gel electrophoresis, which separates fragments by size

• The smallest fragments move farthest in the gel

• These fragments can be visualized when stained with ethidium bromide and illuminated by UV light

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Section 20.4

• A Southern blot is used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments from restriction digest

• Southern blots can also be used to – determine whether a clone contains all or part of a gene – ascertain the size and sequence organization of a gene or DNA

sequence of interest

• Southern blot has two components: separation of DNA

fragments by gel electrophoresis and hybridization by using labeled probes

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Section 20.4

• Northern blot analysis is used to determine whether a gene is actively being expressed in a given cell or tissue – Used to study patterns of gene expression in embryonic tissues,

cancer, and genetic disorders

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Section 20.4

• Fluorescent in situ hybridization (FISH) involves hybridizing a probe directly to a chromosome or RNA without blotting

• FISH can be carried out with isolated chromosomes on a slide or in situ in tissue sections or entire organisms

• FISH is especially helpful when embryos are used for various studies in developmental genetics

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20.5 DNA Sequencing Is the Ultimate Way to Characterize DNA Structure at the

Molecular Level

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Section 20.5

• The most common method of DNA sequencing is dideoxynucleotide chain- termination sequencing (Sanger sequencing) developed by Sanger

• This technique involves the addition of a small amount of dideoxynucleotide, which causes DNA synthesis to terminate

• Large-scale genome sequencing is automated and uses fluorescent dye-labeled dideoxynucleotides

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Section 20.5

• Since the early 1990s, DNA sequencing has largely been done through computer-automated Sanger reaction-based technology (computer-automated high-throughput DNA sequencing) – Generates large amounts of sequence DNA – Enabled the rapid progress of the Human Genome Project

• Large-scale genome sequencing is automated and uses fluorescent dye-labeled dideoxynucleotides

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Section 20.5

• The Sanger technique is outdated when it comes to sequencing entire genomes

• Next-generation sequencing (NGS) technologies will allow faster and cheaper genomic sequencing to take place

• Through 2006, new sequencing technologies were cutting sequencing costs in half about every two years

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next-generation sequencing

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