Introduction to Cloning and Recombinant DNA Technology

Introduction to Cloning and Recombinant DNA Technology David Bedwell, Ph.D. Department of Microbiology Office telephone: 934-6593 Email: dbedwell@uab.edu The Powerpoint slides for this lecture are available for download at: http://www.microbio.uab.edu/bedwell/index4.html Reference: Molecular Biology of the Cell, 5th Edition, by Alberts et al., published by Garland Science, 2008.

Introduction to Cloning and Recombinant Technology: Lecture Outline • Background • DNA cloning • DNA sequencing • Detection of disease genes • Polymerase chain reaction (PCR) • PCR basics • PCR in medicine • PCR in forensics

Plasmid DNA is the genetic material of most organisms (from bacteria to humans) Chromosome: Most bacteria have one circular DNA chromosome ranging in size from 1,000 to 8,000 kilobase pairs. Plasmid: Extrachromosomal genetic element also made of a circular DNA molecule. Bacterial Genome: The collection of all of the genes present on the bacteria’s chromosome or its extrachromosomal genetic elements.

Basics: Nucleotides are the building blocks of DNA Only in RNA, not DNA

Deoxyribonucleic acid (DNA) is a long double-stranded chain of nucleotides • DNA is the hereditary material passed on from generation to generation. • DNA is made up of four nucleotides: A, C, G, and T. • A always pairs with T. • C always pairs with G. • The two strands of DNA are in an antiparallel configuration. • Two complementary DNA strands will separate when heated, and will spontaneously pair together again (hybridize) when cooled.

DNA Double Helix

What Does It Mean: “To Clone”? Clone: a collection of molecules or cells, all identical to an original molecule or cell • To "clone a gene" is to make many copies of it - for example, by replicating it in a culture of bacteria. • Cloned gene can be a normal copy of a gene (= “wild type”). • Cloned gene can be an altered version of a gene (= “mutant”). • Recombinant DNA technology makes manipulating genes possible.

Restriction Enzymes • Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes”. • Cut up “foreign” DNA that invades the cell. • Type II and III restriction enzymes cleave DNA chains at selected sites. • Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage. • An enzyme that recognizes a 6-base sequence is called a "six-base cutter”.

Basics of type II Restriction Enzymes • No ATP requirement. • Recognition sites in double stranded DNA have a 2-fold axis of symmetry – a “palindrome”. • Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends.

Recognition/Cleavage Sites of Type II Restriction Enzymes Examples of Palindromes: Don't nod Dogma: I am God Never odd or even Too bad – I hid a boot Rats live on no evil star No trace; not one carton Was it Eliot's toilet I saw? Murder for a jar of red rum Some men interpret nine memos Campus Motto: Bottoms up, Mac Go deliver a dare, vile dog! Madam, in Eden I'm Adam Oozy rat in a sanitary zoo Ah, Satan sees Natasha Lisa Bonet ate no basil Do geese see God? God saw I was dog Dennis sinned Cuts usually occurs at a palindromic sequence SmaI: produces blunt ends 5´ CCCGGG 3´ 3´ GGGCCC 5´ EcoRI: produces sticky ends 5´ GAATTC 3´ 3´ CTTAAG 5´

Type II restriction enzyme nomenclature Why the funny names? • EcoRI – Escherichia coli strain R, 1st enzyme • BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme • DpnI – Diplococcus pneumoniae, 1st enzyme • HindIII – Haemophilus influenzae, strain D, 3rd enzyme • BglII – Bacillus globigii, 2nd enzyme • PstI – Providencia stuartii 164, 1st enzyme • Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme • KpnI – Klebsiella pneumoniae, 1st enzyme

Results of Type II Digestion • Enzymes with staggered cuts  complementary ends • HindIII - leaves 5´ overhangs (“sticky”) 5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’ 3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’ • KpnI leaves 3´ overhangs (“sticky”) 5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’ 3’--CCATGG-- 5’ 3’ –C CATGG-- 5’

Results of Type II Digestion • Enzymes that cut at same position on both strands leave “blunt” ends • SmaI 5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’ 3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’

Restriction Endonucleases Cleave DNA at specific DNA sequences

DNA Ligase joins DNA fragments together • Enzymes that cut with staggered cuts result in complementary ends that can be ligated together. • HindIII - leaves 5’ overhangs (“sticky”) 5’ --A AGCTT--3’ 5’ --AAGCTT-- 3’ 3’ --TTCGA A--5’ 3’ --TTCGAA-- 5’ • Sticky ends that are complementary (from digests with the same or different enzymes) can be ligated together. • Sticky ends that are not complementary cannot be ligated together.

DNA Ligase can also join blunt ends DNA fragments with blunt ends generated by different enzymes can be ligated together (with lower efficiency), but usually cannot be re-cut by either original restriction enzyme. • SmaI -CCC GGG- • DraI -AAA TTT- • Ligations that re-constitute a SmaI or DraI site (CCCGGG or AAATTT) can be re-cut by SmaI or DraI. • Mixed ligation products (CCCTTT + AAAGGG) cannot be re-cut by SmaI or DraI. -CCCGGG- -AAATTT- -CCCTTT- -AAAGGG-

Any Complementary Ends Can be Ligated • BamHI -G GATCC- -CCTAG G- • BglII -A GATCT- -TCTAG A- • Result -GGATCT- -CCTAGA- No longer palindromic, so not cut by BamHI or BglII

Ampr Ori pBR3224361bp Tetr LacZ MCS pUC18 Ori Ampr Plasmids – vehicles for cloning • Plasmids are naturally occurring extrachromosomal DNA molecules. • Plasmids are circular, double-stranded DNA. • Plasmids are the means by which antibiotic resistance is often transferred from one bacteria to another. • Plasmids can be cleaved by restriction enzymes, leaving sticky or blunt ends. • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid.

Ampr Ori pBR3224361bp Tetr LacZ MCS pUC18 Ori Ampr Cloning Vectors Older cloning vector • A cloning vector is a plasmid that can be modified to carry new genes. • Plasmids useful as cloning vectors must have: • An origin of replication. • A selectable marker (antibiotic resistance gene, such as ampr and tetr). • Multiple cloning site (MCS) (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers). • Easy to purify away from host DNA. Newer cloning vector

CFTR LacZ MCS pUC18-hCFTR Ori Ampr Chimeric Plasmids • Named for mythological beast (chimera) with body parts from several creatures. • After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted. • Ends of the plasmid/fragment are closed to form a "recombinant plasmid”. • Plasmid can replicate when placed in a suitable bacterial host.

DNA cloning requires restriction endonuclease and DNA ligase • Consider a plasmid with a unique EcoRI site: • 5' NNNNGAATTCNNNN 3' • 3’ NNNNCTTAAGNNNN 5' • An EcoRI restriction fragment of foreign DNA can be inserted into a plasmid having an EcoRI cloning site by: • a) cutting the plasmid at this site with EcoRI, • b) annealing the linearized plasmid with the EcoRI foreign DNA fragment, and, • c) sealing the nicks with DNA ligase. • 5' NNNNGAATTCNNNN 3' • 3' NNNNCTTAAGNNNN 5’ • This results in a recombinant DNA molecule.

Key features of DNA replication are used in DNA sequencing • DNA synthesis occurs in the 5´ to 3´ direction. • DNA synthesis requires a template and a primer. • DNA replication is semi-conservative (one strand copied). • DNA replication is carried out by an enzyme called DNA polymerase.

DNA synthesis requires a 3´-OH to make the next phosphodiester bond during DNA synthesis normal dNTP

Dideoxy NTPs block DNA synthesis H

ddNTPs block formation of the next phosphodiester bond during DNA synthesis A 3´-OH on the last ribose is needed for DNA synthesis ddNTP H H A nucleotide-specific stop in DNA synthesis

A mixture of dNTPs and ddNTPs are used in DNA sequencing

Polyacrylamide gel electrophoresis is used to visualize the results of the sequencing reaction

Automated DNA sequencing with fluorescent dyes coupled to each reaction Fluorescent dye coupled to reaction allows visualization of di-deoxy termination events by means of a laser that detects the colored product. This shows four different reactions as done with the old manual sequencing.

Automated DNA sequencing output-4 reactions carried out in one tube

Molecular Medicine: The Human Genome Project 3.2x109 nucleotide pairs NCBI.nlm.nih.gov/genome/guide/human/index

Technology now exists to sequence everyone’s DNA Took just 4 months, $1.5 million to obtain the entire DNA sequence of James Watson.

The genomes of many organisms have been sequenced

Genome resources for many organisms are available

Understanding the arrangement of genes may help understand disease

Southern blot: One way to detect genome structure and disease markers in genomic DNA -Purify genomic DNA -Digest with restriction enzyme -Run agarose gel

Restriction fragment length polymorphisms (RFLPs) can be associated with disease alleles Southern Blot Consider two alleles of a gene. Allele A has 3 BamHI sites, while allele a has only two BamHI sites. probe HpaI Digest Variants 1 2 3 Nor- mal 70% of carriers of the sickle cell gene have a 13.0 kb HpaI fragment. 30% of carriers have 7.0 kb HpaI fragment

Direct Detection of a Sickle Cell Mutation by RFLP A specific hemoglobin mutation Wild TypeMutant Pro Glu Pro Val CCT GAG CCT GTG DdeI site no DdeI site [DdeI cuts at CTNAG] AS AS SS AA Gene encoding sickle cell b-subunit Gene encoding Wild type b-subunit

Polymerase Chain Reaction (PCR) • Allows quick identification of genetic markers: • Identify bacteria in infections • Identify viruses in virus infections • Paternity testing, genetic counseling, forensics • Can exclude individuals, but cannot prove guilt. • Requires only small amounts of DNA. • A repetitive DNA synthesis reaction. • Thermostable DNA polymerase: • Isolated from bacteria in hot springs or near thermal vents in the deep ocean. • Requires gene-specific DNA primers and deoxyribonucleotide triphosphates (dNTPs).

Polymerase Chain Reaction (PCR) A thermophilic (heat-loving) bacteria called Thermus aquaticus is the source of Taq DNA polymerase used in PCR reactions.

The first round of PCR 94°C 37-65°C 70-75°C

PCR increases the yield of DNA exponentially

A typical PCR protocol • Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence. • Next, denature the DNA to single strands at 94˚C. • Rapidly cool the DNA (37-65˚C) and anneal primers to complementary single strand sequences flanking the target DNA. • Extend primers at 70-75˚C using a heat-resistant DNA polymerase such as Taq polymerase derived from Thermus aquaticus. • Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220) to 35 trillion copies (245) of the target DNA. • Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely. • Cool to 4˚C and store or use amplified PCR product for analysis.

5´ 3´ 3´ 5’ A 5’ 3´ A B 3´ 5´ A B 5´ 3´ B 3´ 5´ PCR cycle 28 – ~1 billion strands 2 original strands. 28 strands starting with primer A, but with no end. 28 strands starting with primer B, but with no end. ~500,000,000 strands starting with primers A (5´) and ending with primer B (referred to as unit-length strand in previous figure). ~500,000,000 strands starting with primer B (5´) and ending with primer A (referred to as unit-length strand in previous figure).

PCR in Medicine • Since 1987, PCR has had a major impact on prenatal diagnosis of single gene disorders. • Also very important in carrier testing for genetic diseases. • Improved speed, accuracy and technical flexibility over previous methods.

PCR and prenatal diagnosis • For prenatal diagnosis, PCR used to amplify DNA from fetal cells obtained from amniotic fluid. • Single base changes then detected by one or more of following: -dot blot (spot hybridization) with oligonucleotides specific for known mutation. -restriction enzyme analysis (RFLP). -direct sequencing of DNA. • Important to be certain of result so combination of two methods provides confirmation. • Many other conditions can be detected with same approach, including: -Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia, Huntingdon's disease, Duchenne muscular dystrophy (DMD).

Introduction to Cloning and Recombinant DNA Technology