Recombinant DNA Technology

This achievement ushered in the era of gene cloning Recombinant DNA technology is the use of in vitro molecular techniques to isolate and manipulate fragments of DNA In the early 1970s, researchers at Stanford University were able to construct chimeric molecules called recombinant DNA molecules Shortly thereafter, it became possible to introduce such molecules into living cells where they replicated to make many identical copies. This achievement ushered in the era of gene cloning Recombinant DNA technology and gene cloning have been fundamental to our understanding of gene structure and function Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Cloning Experiments Involve Chromosomal and Vector DNA Cloning experiments usually involve two kinds of DNA molecules Chromosomal DNA Serves as the source of the DNA segment of interest Vector DNA Serves as the carrier of the DNA segment that is to be cloned Can replicate independently of the host chromosomal DNA To prepare chromosomal DNA, the scientist has to Obtain cellular tissue from the organism of interest Break open the cells Extract and purify DNA using a variety of biochemical techniques Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The cell that harbors the vector is called the host cell When a vector is replicated inside a host cell, the DNA that it carries is also replicated The sequence of the origin of replication determines whether that vector can replicate in a particular host cell The vectors commonly used in gene cloning were originally derived from two natural sources 1. Plasmids 2. Viruses Many naturally occurring plasmids have selectable markers Typically, genes conferring antibiotic resistance to the host cell Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Cloning Experiments Involve Enzymes that Cut and Paste DNA Insertion of chromosomal DNA into a vector requires the cutting and pasting of DNA fragments The enzymes used to cut DNA are known as restriction endonucleases or restriction enzymes These bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Restriction enzymes bind to specific DNA sequences These are typically palindromic The sequence is identical when read in the opposite direction in the complementary strand For example, the EcoRI recognition sequence is 5’ GAATTC 3’ 3’ CTTAAG 5’ Some restriction enzymes digest DNA into fragments with “sticky ends” These DNA fragments will hydrogen bond to each other due to their complementary sequences Other restriction enzymes generate blunt ends The enzyme NaeI Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Cleavage by restriction enzymes is the first step to making recombinant DNA. In this case, the ends are ‘sticky’ in that they have a short, single-stranded end that can base-pair with another piece of DNA cut with the same enzyme.

Restriction enzymes are made naturally by many species of bacteria Restriction enzymes were discovered in the 1960s and 1970s by Werner Arber, Hamilton Smith and Daniel Nathans Restriction enzymes are made naturally by many species of bacteria They protect bacterial cells from invasion by foreign DNA, particularly that of bacteriophage Currently, several hundred different restriction enzymes are available commercially Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

A recombinant DNA molecule This interaction is not stable because it involves only a few hydrogen bonds To establish a permanent connection, the sugar-phosphate backbones of the two DNA fragments must be covalently linked A recombinant DNA molecule

The Steps in Gene Cloning The procedure shown seeks to clone the human b-globin gene into a plasmid vector The vector carries two important genes ampR  Confers antibiotic resistance to the host cell Identifies cells that have taken up the vector lacZ  Encodes b-galactosidase Provides a means by which bacteria that have picked up the cloned gene can be identified Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

This is termed a hybrid vector Digestion of a human cell would actually produce tens of thousands of fragments. This is termed a hybrid vector

Cells that are able to take up DNA are called competent cells This step of the procedure is termed transformation, when plasmid vectors are used, and transfection, when a viral vector is introduced into a host cell Cells that are able to take up DNA are called competent cells X-Gal is an analog of lactose IPTG – molecular mimic of allolactose Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

All bacterial colonies growing on the plate had to have picked up the vector and its ampR gene But how to differentiate between the colonies that have a circularized vector from those with a hybrid vector? In the hybrid vector, the chromosomal DNA inserts into the lacZ gene, thereby disrupting it By comparison, the recircularized vector has a functional lacZ gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The growth media contains two relevant compounds: IPTG (isopropyl-b-D-thiogalactopyranoside) A lactose analogue that can induce the lacZ gene X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside) A colorless compound that is cleaved by b-galactosidase into a blue dye The color of bacterial colonies will therefore depend on whether or not the b-galactosidase is functional If it is, the colonies will be blue If not, the colonies will be white In this experiment Bacterial colonies with recircularized vectors form blue colonies While those with hybrid vectors form white colonies Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The net result of gene cloning is to produce an enormous amount of copies of a gene During transformation, a single bacterial cell usually takes up a single copy of the hybrid vector Amplification of the gene occurs in two ways: 1. The vector gets replicated by the host cell many times This will generate a lot of copies per cell (25-50 for plasmids) 2. The bacterial cell divides approximately every 20 minutes This will generate a population of many million overnight Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

cDNA To clone DNA, one can start with a sample of RNA The enzyme reverse transcriptase is used Uses RNA as a template to make a complementary strand of DNA From retroviruses to copy their RNA genome to DNA DNA that is made from RNA is called complementary DNA (cDNA) It could be single- or double-stranded Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

polyA tail

This has two ramifications From a research perspective, an important advantage of cDNA is that it lacks introns This has two ramifications 1. It allows researchers to focus their attention on the coding sequence of a gene 2. It allows the expression of the encoded protein Especially, in cells that would not splice out the introns properly (e.g., a bacterial cell) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Polymerase Chain Reaction Another way to copy DNA is a technique called polymerase chain reaction (PCR) It was developed by Kary Mullis in 1985 Unlike gene cloning, PCR can copy DNA without the aid of vectors and host cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Chromosomal DNA Gene of interest Primer binding near one end of the gene A different primer binding near the other end of the gene Many PCR cycles Many copies of the gene of interest, flanked by the regions where the primers bind. (a) The outcome of a PCR experiment Template DNA Site where reverse primer binds 5′ 3′ 3′ 5′ Site where forward primer binds Denaturation: Separate DNA strands with high temperature. 5′ 3′ 3′ 5′ Primer annealing: Lower temperature, which allows primers to bind to template DNA. 5′ 3′ Forward primer 3′ 5′ 5′ 3′ Reverse primer 5′ 3′ 3′ 5′ T G C A C C A G C A T C C T C A C G T G G T C G T A G G G A C T A G Primer extension: Incubate at a temperature that allows DNA synthesis to occur. 3′ 5′ Reverse primer 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ (b) The 3 steps of a PCR cycle Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The starting material for PCR includes 1. Template DNA Contains the region that needs to be amplified 2. Oligonucleotide primers Complementary to sequences at the ends of the DNA fragment to be amplified These are synthetic and about 15-20 nucleotides long 3. Deoxynucleoside triphosphates (dNTPs) Provide the precursors for DNA synthesis 4. Taq polymerase DNA polymerase isolated from the bacterium Thermus aquaticus This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Binding of the primers to the DNA is called annealing PCR is carried out in a thermocycler, which automates the timing of each cycle All the ingredients are placed in one tube The experimenter sets the machine to operate within a defined temperature range and number of cycles Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

A typical PCR run is likely to involve 20 to 30 cycles of replication The sequential process of denaturing-annealing-synthesis is then repeated for many cycles A typical PCR run is likely to involve 20 to 30 cycles of replication This takes a few hours to complete After 20 cycles, a DNA sample will increase 220-fold (~ 1 million-fold) After 30 cycles, a DNA sample will increase 230-fold (~ 1 billion-fold) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

RT-PCR is carried out in the following manner PCR is also used to detect and quantitate the amount of RNA in living cells The method is called reverse transcriptase PCR (RT-PCR) RT-PCR is carried out in the following manner RNA is isolated from a sample It is mixed with reverse transcriptase and a primer that will anneal to the 3’ end of the RNA of interest This generates a single-stranded cDNA which can be used as template DNA in conventional PCR RT-PCR is extraordinarily sensitive It can detect the expression of small amounts of RNA in a single cell Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3′ 3′ 5′ RNA isolated from a sample of cells 3′ 5′ 3′ 5’ ′ 5′ 5′ 3′ RNA of interest Add reverse transcriptase, a primer that binds near the 3′ of the RNA of interest, and deoxyribonucleotides 3′ 3′ 5′ 3′ 5′ 3′ 5′ 5′ 3′ 5′ 3′ 5′ Subject to PCR as described in Figures 18.5 and 18.6 Primer Double-stranded cDNAs derived from the RNA of interest Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

DNA LIBRARIES AND BLOTTING METHODS Molecular geneticists usually want to study particular genes within the chromosomes of living species This presents a problem, because chromosomal DNA contains thousands of different genes The term gene detection refers to methods that distinguish one particular gene from a mixture of thousands of genes Scientists have also developed techniques to identify gene products RNA that is transcribed from a particular gene Protein that is encoded in an mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

DNA Libraries A DNA library is a collection of thousands of different cloned fragments of DNA When the starting material is chromosomal DNA, the library is called a genomic library A cDNA library contains hybrid vectors with cDNA inserts Should represent the genes expressed in the cells the RNA was isolated from Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Copyright ©The McGraw-Hill Companies, Inc Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

In most cloning experiments, the ultimate goal is to clone a specific gene For example, suppose that a geneticist wishes to clone the rat b-globin gene Only a small percentage of the hybrid vectors in a DNA library would actually contain the gene Therefore, geneticists must have a way to distinguish those rare colonies from all the others This can be accomplished by using a DNA probe in a procedure called colony hybridization Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

But how does one obtain the probe? If the gene of interest has been already cloned, a piece of it can be used as the probe If not, one strategy is to use a probe that likely has a sequence similar to the gene of interest For example, use the rat b-globin gene to probe for the b-globin gene from another rodent What if a scientist is looking for a novel type of gene that no one else has ever cloned from any species? If the protein of interest has been previously isolated, amino acid sequences are obtained from it The researcher can use these amino sequences to design short DNA probes that can bind to the protein’s coding sequence Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Stained DNA gel under UV

Southern Blotting Southern blotting can detect the presence of a particular gene sequence within a mixture of many It was developed by E. M. Southern in 1975 Southern blotting has several uses 1. It can determine copy number of a gene in a genome 2. It can detect small gene deletions that cannot be detected by light microscopy 3. It can identify gene families 4. It can identify homologous genes among different species 5. It can determine if a transgenic organism is carrying a new or modified gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Prior to a Southern blotting experiment, the gene of interest, or a fragment of a gene, has been cloned This cloned DNA is labeled (e.g., radiolabeled) and used as a probe The probe will be able to detect the gene of interest within a mixture of many DNA fragments Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Gel electrophoresis of total DNA digested with restriction enzyme and subjected to Southern blot hybridization

Northern Blotting Northern blotting is used to identify a specific RNA within a mixture of many RNA molecules It was not named after anyone called Northern! Originally known as ‘Reverse-Southern’ which became Northern. Northern blotting has several uses 1. It can determine if a specific gene is transcribed in a particular cell type Nerve vs. muscle cells 2. It can determine if a specific gene is transcribed at a particular stage of development Fetal vs. adult cells 3. It can reveal if a pre-mRNA is alternatively spliced Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Northern blotting is rather similar to Southern blotting It is carried out in the following manner RNA is extracted from the cell(s) and purified It is separated by gel electrophoresis It is then blotted onto nitrocellulose or nylon filters The filters are placed into a solution containing a radioactive probe The filters are then exposed to an X-ray film RNAs that are complementary to the radiolabeled probe are detected as dark bands on the X-ray film Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The three mRNAs have different molecular weights Smooth and striated muscles produce a larger amount of tropomyosin mRNA than do brain cells This is expected because tropomyosin plays a role in muscle contraction The three mRNAs have different molecular weights This indicates that the pre-mRNA is alternatively spliced Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Western Blotting Western blotting is used to identify a specific protein within a mixture of many protein molecules Again, it was not named after anyone called Western! Western blotting has several uses 1. It can determine if a specific protein is made in a particular cell type Red blood cells vs. brain cells 2. It can determine if a specific protein is made at a particular stage of development Fetal vs. adult cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Western blotting is carried out as follows: Proteins are extracted from the cell(s) and purified They are then separated by SDS-PAGE They are first dissolved in the detergent sodium dodecyl sulfate This denatures proteins and coats them with negative charges The negatively charged proteins are then separated by polyacrylamide gel electrophoresis They are then blotted onto nitrocellulose or nylon filters The filters are placed into a solution containing a primary antibody (recognizes the protein of interest) A secondary antibody, which recognizes the constant region of the primary antibody, is then added The secondary antibody is also conjugated to alkaline phosphatase The colorless dye XP is added Alkaline phosphatase converts the dye to a black compound Thus proteins of interest are indicated by dark bands

Western blot analysis

Western blot analysis

This experiment indicates that b-globin is made in red blood cells but not in brain or intestinal cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Techniques that Detect the Binding of Proteins to DNA or RNA Researchers often want to study the binding of proteins to specific sites on a DNA or RNA molecule For example, the binding to DNA of transcription factors To study protein-DNA interactions, the following two methods are used 1. Gel retardation assay Also termed band shift assay 2. DNA footprinting Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The technical basis for a gel retardation assay is this: The binding of a protein to a fragment of DNA retards its rate of movement through a gel Higher mass and therefore slow migration Lower mass and therefore fast migration Gel retardation assays must be performed under nondenaturing conditions Buffer and gel should not cause the unfolding of the proteins nor the separation of the double helix Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

DNA Sequencing During the 1970s two DNA sequencing methods were devised One method, developed by Alan Maxam and Walter Gilbert, involves the base-specific cleavage of DNA The other method, developed by Frederick Sanger, is known as dideoxy sequencing The dideoxy method has become the more popular and will therefore be discussed here Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The dideoxy method is based on our knowledge of DNA replication but uses a clever twist DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’–P of one and the 3’–OH of the other. Nucleotides missing that 3’–OH can be synthesized Sanger reasoned that if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow This is referred to as chain termination If ddATP is used, termination will always be at an A in the DNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

This is accomplished using cloning or PCR techniques Prior to DNA sequencing, the DNA to be sequenced must be obtained in large amounts This is accomplished using cloning or PCR techniques In many sequencing experiments, the target DNA is cloned into the vector at a site adjacent to a primer annealing site Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Copyright © The McGraw-Hill Companies, Inc Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A G G C T T C C Sequence to be analyzed (target DNA) G T T Primer G A The newly-made DNA fragments can be separated according to their length by running them on an acrylamide gel Annealing site C 5′ Recombinant vector They can then be visualized as fluorescence peaks as the bands run off the bottom of the gel Many copies of the recombinant vector, primer, dNTPs, fluorescently labeled dideoxynucleotides, and DNA polymerase are mixed together. Incubate to allow the synthesis of DNA. CACCGTAAGGACTddG CACCGTAAGGACddT CACCGTAAGGAddC CACCGTAAGGddA CACCGTAAGddG CACCGTAAddG CACCGTAddA CACCGTddA CACCGddT CACCddG CACddC CAddC CddA ddC Nucleotides added to primer Separate newly made strands by gel electrophoresis. Sequence deduced from gel G T C A C A C C G T A A G G A C T G G G A A T G C C Laser beam A C Fluorescence detector (a) Automated DNA sequencing (b) Output from automated sequencing

An important innovation in the method of dideoxy sequencing is automated sequencing It uses a single tube containing all four dideoxyribonucleotides However, each type (ddA, ddT, ddG, and ddC) has a different-colored fluorescent label attached After incubation and polymerization, the sample is loaded into a single lane of a gel Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The procedure is automated using a laser and fluorescent detector The fragments are separated by gel electrophoresis Indeed, the mixture of DNA fragments are electrophoresed off the end of the gel As each band comes off the bottom of the gel, the fluorescent dye is excited by the laser The fluorescence emission is recorded by the fluorescence detector The detector reads the level of fluorescence at four wavelengths