Recombinant DNA Technology and Molecular Cloning Chapter 8: Recombinant DNA Technology and Molecular Cloning

Sometimes a good idea comes to you when you are not looking for it Sometimes a good idea comes to you when you are not looking for it. Through an improbable combination of coincidences, naiveté and lucky mistakes, such a revelation came to me one Friday night in April, 1983, as I gripped the steering wheel of my car and snaked along a moonlit mountain road into northern California’s redwood country. That was how I stumbled across a process that could make unlimited numbers of copies of genes, a process now known as the polymerase chain reaction (PCR) Kary B. Mullis, Scientific American (1990), 262:36

8.1 Introduction

The cornerstone of most molecular biology technologies is the gene. To facilitate the study of a genes: Clone the gene by inserting it into another DNA molecule that serves as a vehicle or vector that can be replicated in living cells.

When two DNAs (the insert and vector) of different origin are combined, the result is a recombinant DNA molecule. The recombinant DNA is placed in a host cell, amplified, and purified for further analysis.

8.2 The beginnings of recombinant DNA technology

Recombinant DNA technology arose through the efforts of several research groups working primarily on bacteriophage lambda ().

Insights from bacteriophage lambda () cohesive sites In 1962, Allan Campbell noted that the linear genome of bacteriophage  forms a circle upon entering the host bacterial cell by joining complementary single-stranded DNA cohesive (cos) sites. The idea of joining DNA segments by “cohesive sites” became a guiding principle in the development of recombinant DNA technology.

Insights from bacterial modification and restriction systems Salvador Luria and other phage workers made the following observations: Phages grown in one bacterial host fail to grow in a different “restrictive” bacterial host. The phage DNA is degraded in the “restrictive” host.

Rare progeny phages become “modified” in some way so that they grow normally in the new host. The modification was reversible. 1962: The molecular basis of restriction and modification was defined by Werner Arber and coworkers.

Restriction system Restriction endonucleases First restriction endonuclease characterized in E. coli K-12 by Matt Meselson and Bob Yuan. “Restrict” or prevent viral infection by degrading the invading nucleic acid.

Modification system Methylase activity: Addition of methyl groups to protect those sites in DNA sensitive to attack by a restriction endonuclease. Typically adenine methylation (6-methyl adenine). Methylation pattern is maintained during DNA replication.

The first cloning experiments One of the first recombinant DNA molecules was a hybrid of phage and the SV40 mammalian DNA virus genome. 1974: first eukaryotic gene was cloned. Amplified ribosomal RNA (rRNA) genes from Xenopus laevis oocytes were cloned into a bacterial plasmid. The cloned frog genes were actively transcribed into rRNA in E. coli.

I was tempted then to put together a book called the Whole Risk Catalogue. It would contain risks for old people and young people and so on. It would be a very popular book in our semi-paranoid society. Under “D” I would put dynamite, dogs, doctors, dieldrin [an insecticide] and DNA. I must confess to being more frightened of dogs. But everyone has their own things to worry about. James Watson, Genetics and Society (1993)

Fear of recombinant DNA molecules 1975: Recommendations from a landmark meeting of molecular biologists formed the basis for official guidelines developed by the National Institutes of Health (NIH). Activities involving the handling of recombinant DNA and organisms must be conducted in accordance with the NIH guidelines. Four levels of risk are recognized, from minimal to high.

8.3 Cutting and joining DNA

Two main categories of enzymes are important tools in the preparation of recombinant DNA DNA ligases: join two pieces of DNA by forming phosphodiester bonds. Restriction endonucleases: recognize a specific, rather short, nucleotide sequence on a double-stranded DNA molecule, called a restriction site, and cleave the DNA at this site or elsewhere.

Major classes of restriction endonucleases Type II restriction endonucleases are widely used by molecular biologists. >240 available commercially. 6 bp cutters are the most commonly used.

Restriction endonucleases are named for the organism in which they were discovered: HindIII from Haemophilus influenza (strain d) SmaI from Serratia marcescens EcoRI from Escherichia coli (strain R) BamHI from Bacillus amyloliquefaciens (strain H)

Recognition sequences for type II restriction endonucleases Orthodox type II restriction endonucleases function as homodimers. Recognition sequences are typically palindromes. Some enzymes generate “sticky ends.” Some enzymes generate “blunt ends.”

Restriction endonucleases exhibit a great degree of sequence specificity. A single base pair change in the recognition site eliminates enzymatic activity.

The steps involved in restriction endonuclease DNA binding and cleavage The first contact is nonspecific binding: Interaction with the DNA sugar-phosphate backbone only. Catalytic center kept at a safe distance.

“Hopping” or “jumping” over longer distances. Random walk: “Sliding” over short distances of <30-50 bp to target restriction site. “Hopping” or “jumping” over longer distances. Specific binding at restriction site: Large conformational change of the enzyme and DNA (coupling). Activation of catalytic center.

EcoRI: kinking and cutting DNA Common structural core of four conserved -strands and one -helix. Large conformational change in EcoRI and the DNA upon specific binding. A central kink in the DNA brings the critical phosphodiester bond between G and A deeper into the active site and unwinds the DNA.

In the presence of Mg2+, EcoRI cleaves the DNA on both strands at the same time to give free 5′-phosphate and 3′-OH ends. The exact mechanism by which cleavage occurs has not yet been proven experimentally.

DNA ligase joins linear pieces of DNA The DNA ligase most widely used in the lab is from bacteriophage T4. T4 DNA ligase catalyzes formation of a phosphodiester bond between the 5′-phosphate of a nucleotide on one fragment of DNA and the 3′-hydroxyl of another.

T4 DNA ligase will ligate fragments with sticky ends or blunt ends, but for blunt ends the reaction is less efficient. To increase the efficiency of ligation, researchers often use the enzyme terminal deoxynucleotidyl transferase to modify the blunt ends.

8.4 Molecular cloning

Basic molecular cloning procedure DNA fragments to be cloned are generated using restriction endonucleases. Fragments are ligated to other DNA molecules that serve as vectors. Recombinant DNA molecules are transferred to a host cell. Cloned recombinant DNA is recovered from the host cell for analysis.

Choice of vector is dependent on insert size and application Cloning vectors are carrier DNA molecules with four important features: Replicate independently. Contain a number of restriction endonuclease cleavage sites that are present only once. Carry a selectable marker. Relatively easy to recover from host cell.

The greatest variety of cloning vectors has been developed for use in E. coli. The first practical skill generally required by a molecular biologist is the ability to grow pure cultures of bacteria.

Classic cloning vectors: Plasmids Phages Cosmids New generation vectors: Bacterial artificial chromosomes (BACs) Yeast artificial chromosomes (YACs) Mammalian artificial chromosomes (MACs)

Plasmid DNA as a vector Plasmids are named with a system of uppercase letters and numbers, where the lowercase “p” stands for “plasmid.” Low copy number plasmids: replicate to yield only one or two copies in each bacterial cell. High copy number plasmids: replicate to yield >500 copies per bacterial cell.

Plasmid vectors are modified from naturally occurring plasmids Contain a specific antibiotic resistance gene. Contain a multiple cloning site.

Five major steps for molecular cloning using a plasmid vector Construction of a recombinant DNA molecule. Transfer of ligation reaction products to host bacteria. Multiplication of plasmid DNA molecules. Division of host cells and selection of recombinant clones, e.g. by blue-white screening. Amplification and purification of recombinant plasmid DNA.

Transformation: transfer of recombinant plasmid DNA to a bacterial host Bacterial cells are incubated in a concentrated calcium salt solution to make their membranes leaky. The permeable “competent” cells are mixed with DNA to allow DNA entry. Alternatively, a process called electroporation drives DNA into cells by a strong electric current.

Why isn’t the introduced foreign plasmid DNA degraded by a bacterial restriction-modification system?

Recombinant selection Antibiotic resistance selects for transformed bacterial cells. Numerous cell divisions of a single transformed bacteria result in a clone of cells visible as a bacterial colony on an agar plate. Successfully transformed bacteria will carry either recombinant or nonrecombinant plasmid DNA.

Blue-white screening In the case of the vector pUC18, blue-white screening is used to distinguish recombinant from nonrecombinant transformants. Also known as “lac selection” or - complementation

-galactosidase activity can be used as an indicator of the presence of foreign DNA If the lacZ 5′ region of pUC18 is not interrupted by inserted foreign DNA, the amino-terminal portion of -galactosidase is synthesized. The mutant E. coli host encodes only the carboxyl end of  -galactosidase.

The N-terminal and C-terminal fragments come together to form a functional enzyme. -galactosidase activity can be measured using a colorless chromogenic substrate called X-gal. Cleavage of X-gal produces a blue-colored product, visualized as a blue colony on an agar plate. If a foreign insert has disrupted the lacZ 5′ coding sequence, X-gal is not cleaved and the bacterial colonies remain white.

Amplification and purification of recombinant plasmid DNA Further screening to confirm the presence and orientation of the insert. Amplify positive (white) colony containing recombinant plasmid DNA in liquid culture. Purify plasmid DNA from crude cell lysates, e.g. by chromatography and ethanol precipitation.

Liquid chromatography Molecules dissolved in a solution will interact (bind and dissociate) with a solid surface. When the solution is allowed to flow across the surface, molecules that interact weakly with the solid surface will spend less time bound to the surface and will move more rapidly. Commonly used to separate mixtures of nucleic acids and proteins.

Three main techniques Gel filtration chromatography: separation by differences in mass. Ion-exchange chromatography: separation by differences in charge. Affinity chromatography: separation by differences in binding affinity.

Bacteriophage lambda () as a vector Phage vectors are particularly useful for preparing genomic libraries. The recombinant viral particle infects bacterial host cells in a process called transduction. Progeny viral particles appears as a clear spot of lysed bacteria or “plaque” on a lawn of bacteria.

Artificial chromosome vectors Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are important tools for mapping and analysis of complex eukaryotic genomes. 1997: first prototype mammalian artificial chromosome (MAC)

Yeast artificial chromosome (YAC) vectors YAC vectors are designed to act like chromosomes in host yeast cells Origin of replication (Autonomously replicating sequence, ARS) Centromere Telomere

YAC vectors contain selectable markers URA3: encodes an enzyme required for uracil biosynthesis. TRP1: encodes an enzyme required for tryptophan biosynthesis. SUP4: tRNA that suppresses the Ade2-1 UAA mutation.

Red-white selection Host yeast strain: ura3/trp1/Ade2-1 mutant When foreign DNA is inserted in the multiple cloning site, SUP4 expression is interrupted. The Ade2-1 mutation is no longer suppressed. ADE1 and ADE2 encode enzymes involved in adenine biosynthesis.

Ade2-1 mutant cells produce a red pigment from polymerization of an intermediate compound. In the absence of foreign DNA, SUP4 is expressed. The Ade2-1 mutation is suppressed. Ade2-1 mutant cells expressing SUP4 are white (the color of wild-type yeast cells).

Sources of DNA for cloning Genomic DNA Chemically synthesized oligonucleotides Previously isolated clones: subcloning Complementary DNA (cDNA) Polymerase chain reaction (PCR)

Complementary DNA (cDNA) synthesis Most eukaryotic mRNAs have a poly(A) tail. The poly(A) region can be used to selectively isolate mRNA from total RNA by affinity chromatography. The purified mRNA can then be used as a template for synthesis of cDNA by reverse transcriptase.

Are 5′→3′ sequences that appear in the literature the “first strand” or “second strand” of the double-stranded cDNA?

Polymerase chain reaction (PCR) Basic requirements for in vitro DNA synthesis: DNA polymerase DNA template Free 3′-OH to get the polymerase started dNTPs

Three steps of the reaction performed in an automated thermal cycler Denaturation of the template DNA (e.g. 95C). Annealing of primers (e.g. 55-65C). Primer extension by a thermostable DNA polymerase (e.g. 72C).

Taq DNA polymerase from Thermus aquaticus is the most popular enzyme. Pfu DNA polymerase from Pyrococcus furiosus has higher fidelity. Are the primers made of RNA or DNA?

Constructing DNA libraries Genomic library: A cloned set of DNA fragments that represent the entire genome of an organism. cDNA library: A cloned set of the coding region of expressed genes only; derived from mRNA isolated from a specific tissue, cell type, or developmental state.

Genomic library Break DNA into manageable sized pieces (e.g. 15-20 kb for phage  vectors) by partial restriction endonuclease digest. Purify fragments of optimal size by gel electrophoresis or centrifugation techniques. Insert fragments into a suitable vector. For the human genome, approximately 106 clones are required to ensure that every sequence is represented.

cDNA library Does a cDNA library included intron sequences or gene regulatory regions?

8.5 Library screening and probes

Nowadays, a DNA sequence of interest is more likely to be isolated by PCR than by a library screen. In PCR, the pair of primers limits the amplification process to the particular DNA sequence of interest. In contrast, a DNA library can be perpetuated indefinitely in host cells and retrieved whenever the researcher wants to seek out a particular fragment.

A key element required to identify a gene during library screening is the probe: A probe is a nucleic acid (usually DNA) that has the same or a similar sequence to that of a specific gene or DNA sequence of interest. The denatured probe and target DNA can hybridize when they are renatured together.

Library screening involves basic principles of nucleic acid hybridization Double-stranded nucleic acids can undergo denaturation. Complementary single strands spontaneously anneal to a nucleic acid probe to form a hybrid duplex. The nucleic acid probe can detect a complementary molecule in a complex mixture with exquisite sensitivity and specificity.

Types of DNA and RNA probes Oligonucleotide probes: chemically synthesized DNA probes: cloned DNAs RNA probes (riboprobes): made by in vitro transcription from cloned DNA templates

Heterologous probes A probe that is similar to, but not exactly the same as, the nucleic acid sequence of interest.

Homologous probes A probe that is exactly complementary to the nucleic acid sequence of interest. Examples include: Degenerate probes Expressed sequence tag (EST) based probes cDNA probes

Use of degenerate probes: historical perspective Before the advent of genome sequence databases, the classic method for designing a probe relied on having a partial amino acid sequence of a purified protein. Traditionally protein sequencing was performed by Edman degradation. Today protein sequencing is more often performed using mass spectrometry technology.

Unique EST-based probes ESTs are partial cDNA sequences of about 200-400 bp. A computer program applies the genetic code to translate an EST into a partial amino acid sequence. If a match is found with the protein under study, the EST provides the unique DNA sequence of that portion of cDNA. A probe can then be synthesized and used to screen a library for the entire cDNA or genomic clone.

Using an identified cDNA to locate a genomic clone Use of a cDNA to locate a genomic clone provides a highly specific probe for the gene of interest.

Labeling of probes A probe must be labeled, i.e. chemically modified in some way which allows it, and anything it hybridizes to, to be detected.

Radioactive and nonradioactive labeling methods Detection techniques Autoradiography Geiger counter Liquid scintillation counter Phosphorimager

Nonradioactive labeling Colorimetric or chemiluminescent signals Examples: Digoxygenin-conjugated nucleotides are detected with an anti-digoxygenin antibody conjugated to an enzyme or fluorescent dye. Biotin-conjugated nucleotides are detected using enzyme-conjugated streptavidin.

Nucleic acid labeling Method depends on application: Internal (uniform) labeling or end labeling? Radioactive or nonradioactive? Labeling involves DNA or RNA synthesis reactions or other enzyme-mediated reactions.

Some methods for labeling nucleic acids: Random primed labeling In vitro transcription Klenow fill-in

Library screening Five major steps for screening a cDNA library cloned into plasmid vectors: Bacterial colonies are transferred to a nitrocellulose or nylon membrane. Bacterial cells are lysed and DNA is denatured. Labeled probe is added to the membrane. Washed membrane is exposed to X-ray film. Positive colonies are identified.

Transfer of colonies to a DNA-binding membrane Bacterial colonies (members of the library) grown on an agar plate are transferred to nitrocellulose or nylon membrane to make a replica. After lysis and denaturation, the DNA is covalently bound by its sugar-phosphate backbone and the unpaired bases are exposed for complementary base pairing.

Colony hybridization The hybridization step is performed at a nonstringent temperature that ensures the probe will bind to any clone containing a similar sequence. Higher stringency washes are performed to remove nonspecifically bound probe. Heteroduplex stability is influenced by the number of hydrogen bonds between the bases and base stacking hydrophobic interactions.

The shorter the duplex, the lower the GC content, and the more mismatches there are, the lower the melting temperature (Tm). Hybridization temperature is calculated as follows: Tm = 49.82 + 0.41 (%G + C) – (600/l) where l is the length of the hybrid in base pairs.

Detection of positive colonies The resulting autoradiogram has a dark spot on the developed film where DNA-DNA hybrids have formed. If the gene is large, it may be fragmented over multiple clones. The original plate is used to pick bacterial cells with recombinant plasmids that hybridized to the probe.

Screening of expression libraries Expression libraries are made with a cloning vector that contains the required regulatory elements for gene expression. Useful for identifying a clone containing a cDNA of interest when an antibody to the encoded protein is available.

8.6 Restriction mapping and RFLP analysis

Restriction mapping Restriction mapping provides a compilation of the: Number of restriction endonuclease cutting sites along a cloned DNA fragment. Order of restriction endonuclease cutting sites. Distance between restriction endonuclease cutting sites.

Important roles for restriction mapping, for example: Characterizing DNA. Mapping genes. Diagnostic tests for genetic disease. Checking the orientation of the insert in a recombinant DNA clone.

DNA and RNA electrophoresis When charged molecules are placed in an electric field, they migrate toward the positive or negative electrode according to their charge. Nucleic acids are separated by electrophoresis within a matrix or “gel.”

Types of gel electrophoresis Agarose gel electrophoresis Pulsed field gel electrophoresis (PFGE) Polyacrylamide gel electrophoresis (PAGE)

Restriction fragment length polymorphism (RFLP) The existence of alternative alleles associated with restriction fragments that differ in size from each other. Variable regions do not necessarily occur in genes. Function of most RFLPs in the human genome is unknown. Exception: sickle cell anemia RLFP

Diagnosis of sickle cell anemia by restriction fragment length polymorphism (RFLP) and Southern blot A point mutation in the -globin gene has destroyed the recognition site of the restriction endonuclease MstII. Affected individuals: larger restriction fragment on a Southern blot Normal individuals: shorter restriction fragment

RFLPs can serve as markers of genetic disease A RFLP that is close to a disease gene tends to stay with that gene during crossing-over (recombination) during meiosis. Linkage: the likelihood of having one marker transmitted with another through meiosis. When a PCR assay for typing a particular locus is developed, it is generally preferable to RFLP analysis.

Southern blot Method developed by Edward Southern. Identify a specific gene fragment from the often many bands on a gel.

PCR-RFLP assay for maple syrup urine disease Autosomal recessive disease: 1/176 in certain Old Order Mennonite communities. Missense mutation in one of the genes encoding an enzyme involved in metabolism of branched-chain amino acids. Tyrosine (Y) to asparagine (N) substitution: Y393N allele.

Symptoms appear 4 to 7 days after birth. Accumulation of -keto acid derivatives gives urine a maple syrup-like odor. Neurological deterioriation and death within 2 to 3 weeks if diet is not controlled.

PCR-RFLP assay to identify Y393N allele Buccal swab or blood sample. PCR Cut PCR products with ScaI. Agarose gel electrophoresis. Stain with ethidium bromide.

8.7 DNA sequencing

DNA sequencing is the ultimate characterization of a cloned gene. Manual sequencing by the Sanger “dideoxy” DNA method. Automated DNA sequencing. Next-generation sequencing.

Manual sequencing by the Sanger “dideoxy” DNA method Another DNA synthesis reaction… DNA polymerase (T7 DNA polymerase called “Sequenase”) DNA template Free 3′-OH to get the polymerase started dNTPs

If the sequence is “unknown” how is the primer designed? Why is a ddNTP a replication terminator?

Automated DNA sequencing Developed by Leroy Hood and Lloyd Smith in 1986. Each ddNTP terminator is tagged with a different color of fluorophore. DNA samples loaded in a capillary array migrate through a gel matrix by size, from smallest to largest.

Automated DNA sequencing When DNA fragments reach the detection window, a laser beam excites the fluorophores causing them to fluoresce. An electropherogram―a graph of fluorescence intensity versus time―is converted to the DNA sequence by computer software.

Next-generation sequencing The sequencing of spatially separated, clonally amplified DNA templates in a massive array all at the same time. DNA sequences in the range of hundreds of megabases to gigabases can be rapidly obtained.

454 pyrosequencing Individual nucleotides are detected by light production as nascent DNA is synthesized one nucleotide at a time.

Template DNA is prepared by emulsion PCR. The template DNA is immobilized on a bead in a well in the sequencing machine. Solutions of A,C,G, and T nucleotides are sequentially added and removed from the reaction. The enzyme luciferase is used to generate light. Light is only produced when the nucleotide solution complements the first unpaired base of the template.