01. Tools of Molecular Genetics I and II_McDermott_NOTES.pdf

Tools of Genetics [1] TOOLS OF MOLECULAR GENETICS Paul J. McDermott, Ph.D. (843) 792-3462 [email protected] A. TOOLS AND METHODS 1. Restriction Endonucleases 2. Gel Electrophoresis 3. Recombinant DNA 4. Nucleic Acid Hybridization 5. Polymerase Chain Reaction (PCR) 6. DNA Sequencing: Didioxynucleot...

Tools of Genetics [1] TOOLS OF MOLECULAR GENETICS Paul J. McDermott, Ph.D. (843) 792-3462 [email protected] A. TOOLS AND METHODS 1. Restriction Endonucleases 2. Gel Electrophoresis 3. Recombinant DNA 4. Nucleic Acid Hybridization 5. Polymerase Chain Reaction (PCR) 6. DNA Sequencing: Didioxynucleotide Method 7. Massively Parallel (Next Generation) DNA Sequencing B. RECOMBINANT DNA: CLONING METHODS 1. Basic Components 2. Types of Cloning Vectors 3. Plasmid Vectors 4. Cloning Using Plasmid Vectors 5. Cloning Using Bacteriophage Vectors C. TYPES OF LIBRARIES 1. Genomic Libraries 2. cDNA Libraries 3. PCR Libraries D. ANALYSIS OF GENE EXPRESSION 1. Microarrays: Gene Expression Profiling 2. RNAseq Tools of Genetics [2] OBJECTIVES 1. Describe restriction endonucleases and how they cleave DNA at specific sequences. 2. Interpret banding patterns of DNA fragments produced by restriction endonuclease digestion and separated according to size by gel electrophoresis. 3. Explain how restriction endonucleases and DNA ligases are used to create recombinant DNA. 4. Describe the procedures involved in Southern blotting, Northern blotting and Western blotting and the purpose of doing each procedure. 5. Specify the 3 main steps of a PCR cycle. 6. Describe the dideoxynucleotide method of DNA sequencing. 7. Describe the principles of the massively-parallel (next-generation) sequencing method and how the sequence “reads” are interpreted. 8. Explain how bacterial plasmids and bacteriophages are used as cloning vectors. 9. Describe genomic, cDNA and PCR-based libraries and how they are prepared. 10. Specify the types of sequences that would be present in a genomic versus cDNA library. 11. Explain how microarrays are used for gene expression profiling. 12. Describe the steps involved in the RNA-seq method and the types of information about gene expression that can be obtained. Illustrations adapted from: • Basic Medical Biochemistry: A Clinical Approach, © 2009 Lippincott Williams & Wilkins, A Wolters Kluwer business • The Cell: A Molecular Approach © 2000 ASM Press and Sinauer Associates, Inc. • Medical Genetics: An Integrated Approach © 2014 McGraw-Hill Education Tools of Genetics [3] A. TOOLS AND METHODS 1. Restriction Endonucleases: Enzymatic digestion of DNA at a specific sequence (cleavage site). As shown by these examples, the sequence is often a palindrome. Digestion Product Source Cleavage Site EcoRI Escherichia coli RY13 5´ GAATTC 3´ 3´ CTTAAG 5´ 5´ G 3´ CTTAA AATTC 3´ G 5´ BamH1 Bacillus amyloliquefaciens H 5´ GGATCC 3´ 3´ CCTAGG 5´ 5´ G 3´ CCTAG GATCC 3´ 3´overhangs G 5´ Pst1 Providencia stuartii 164 5´ CTGCAG 3´ 3´ GACGTC 5´ 5´ CTGCA 3´ G G 3´ 5´overhangs ACGTC 5´ Sma1 Serratia marcescens Sb 5´ CCCGGG 3´ 3´ GGGCCC 5´ 5´ CCC 3´ GGG AluI Arthrobacter luteus 5´ AGCT 3´ 3´ TCGA 5´ 5´ AG 3´ T C Enzyme GGG 3´ blunt ends C CC 5´ C T 3´ GA 5´ 2. Gel Electrophoresis: Following restriction endonuclease digestion, DNA fragments can be separated according to size by gel electrophoresis. b) Load gel with digested DNA to separate fragments Load digest EcoR1 Sites in λ Phage DNA 5´ 3´ ( ) 48.5 kb 21.2 21.2 kb • DNA fragments migrate in an electrical field toward the positive electrode. • Smaller fragments move faster in the gel and migrate farther. 7.5 7.5 kb 5.6 kb 5.7 5.6 4.9 5.7 kb 4.9 kb 3. Recombinant DNA: DNA ligase DNA Fragment (kb) a) Digest with EcoR1 3.6 3.6 kb (+) Cleave with restriction endonuclease (EcoRI) Compatible ends anneal; Rejoin with DNA ligase Recombinant DNA Tools of Genetics [4] A. TOOLS AND METHODS 4. Nucleic Acid Hybridization a) Southern Blotting: Detection of specific DNA fragments by hybridization to a complementary probe that can be either radioactive or tagged for detection. Genomic DNA 1 • Digest genomic DNA into fragments with restriction endonuclease(s). larger 2• Separate DNA fragments according to size by gel electrophoresis. smaller 3 • Denature DNA into single strands and transfer to a nylon membrane. Because the membrane is positively charged, it has a high binding capacity for DNA. Thus, the DNA strands bind tightly to the nylon membrane. (membrane) 4 • Perform hybridization using a radioactive “probe” complementary to a specific sequence in a DNA fragment bound on the membrane. 5• Use autoradiography to detect the radioactive probe that hybridizes to a specific DNA fragment. membrane Example: Restriction enzyme digestion of genomic DNA followed by Southern blotting 10 kilobases (10 kb) EcoR1 Sma1 BamH1 Pst1 5´ 3´ 3´ 5´ Genomic DNA A B Step 1 - Digest genomic DNA with restriction endonucleases (RE Digests). Step 2 - Run gel electrophoresis followed by Southern blotting to nylon membrane. Step 3 - Hybridization with radioactive probe and autoradiography. Each lane shows expected results of RE digests using probe A or B complementary to the indicated sequence. Probe B Probe A RE Digests: Lane 1: Size standards Lane 2: EcoR1 & BamH1 Lane 3: EcoR1 & Pst1 Lane 4: EcoR1 & Pst1 & BamH1 Lane 5: EcoR1 & Sma1 & BamH1 Digest→ 15 DNA Fragment (kb) DNA Fragment (kb) Digest→ 10 5 2 1 1 2 3 4 Lane 5 15 10 5 2 1 1 2 3 4 Lane 5 Tools of Genetics [5] A. TOOLS AND METHODS 4. Nucleic Acid Hybridization b) Northern Blotting: Detection of specific RNAs 1 • Extract RNA from tissue or cells. 2 • Separate RNA molecules according to size by gel electrophoresis. 3 • Transfer to positively charged nylon membrane and hybridize by using a radioactive probe complementary to a specific sequence in a RNA molecule. c) Western Blotting: Detection of specific proteins • Fractionate proteins according to size by gel electrophoresis. • Transfer to membrane and incubate with an antibody to detect a specific protein. Nota Bene: Protein-antibody binding is not hybridization. I put Western blotting here for the purpose of comparing analytical procedures. Transfer to membrane (bands not visible) Tools of Genetics [6] A. TOOLS AND METHODS 5. Polymerase Chain Reaction (PCR): DNA Amplification by Taq Polymerase (Thermus aquaticus) • PCR utilizes sequence-specific primers to amplify a DNA target sequence. Target DNA sequence Denaturation Heat: 95º C 1. Denaturation (melting) of DNA into single strands by heating Primer annealing 45º to 55º C 2 Annealing of sequence-specific primers to complementary sequences in target DNA 3 Synthesis of target DNA molecules by Taq Polymerase Taq Polymerase 72º C Next Cycle Next Cycle A single PCR cycle consists of 3 steps Denaturation into single-stranded DNA by heating Annealing of sequence-specific primers Synthesis of target DNA sequence (Taq Polymerase) • A PCR run usually consists of 30-40 cycles, depending on the application. Thus, amplification of the target sequence (amplicon) occurs exponentially (230). Because so much target sequence is generated (amplicon), the DNA product is essentially pure. • PCR is an alternative method to cloning for amplifying specific sequences from samples of genomic DNA or generating cDNA sequences from samples of RNA. Nucleic acid sequence from database or amino acid sequence of protein DNA mRNA DNA sample Genomic library cDNA library Reverse Transcriptase Primers cDNA Sequence-specific primers PCR PCR amplification of target sequence DNA or cDNA fragments Primers PCR cDNA fragments Tools of Genetics [7] A. TOOLS AND METHODS 6. DNA Sequencing: Dideoxynucleotide (Sanger) Method a) Main Components of Sequencing Reaction 1 • Target DNA to be sequenced 2 • Complementary sequencing primer 3 • Mixture of dNTPs and fluorescent tagged-ddNTPs 4 • DNA Polymerase 2´,3´-dideoxynucleotide (ddNTP) b) Automated Sequencing by Sanger Method 5´ 3´ 5´ Primer Different size fragments are generated due to termination of DNA Polymerase reaction by incorporation of a ddNTP Separate fragments according to size by gel electrophoresis 3 ´ DNA synthesis in presence of fluorescenttagged chain-terminating dideoxynucleotides Computer Read out Each of the 4 possible bases that terminates a DNA fragment has a different color tag detected by the laser Tools of Genetics [8] A. TOOLS AND METHODS 7. Massively Parallel (Next Generation) DNA Sequencing Next generation sequencing (NGS) technology is revolutionizing genetic medicine, particularly for carrier screening, detecting rare genetic diseases, testing of fetal DNA for chromosomal abnormalities and assessing risks of disease. An entire human genome can be sequenced in a single day for about $1000! There are many variations of next generation sequencing technologies, but the most commonly used technology is referred to as “sequencing by synthesis”. PCR amplification • Instead of separating DNA strands by size, NGS uses positional separation: millions of different template DNA are arranged at unique positions on a flow cell and remain fixed during the entire sequencing reaction. • Each template is extended by a single modified base. A detector resolves both the position of each template on the flow cell and its fluorescent color. • Next, in a step unique to NGS, the modified bases are converted to regular bases, such that they become both extendable and non-fluorescent. This process primes them to undergo subsequent rounds of single base extension and imaging. • At the end of a sequencing run with n imaging cycles, the fluorescence color at each template position in each image is mapped to a base (A, T, C, or G). The bases from a single template position are concatenated to yield a DNA sequence of length n, called a ‘‘read.’’ Reads are are aligned to a reference genome as shown below. Tools of Genetics [9] B. RECOMBINANT DNA: CLONING METHODS 1. Basic Components a) Insert: DNA fragment of interest b) Cloning Vector: DNA molecule capable of autonomous replication in a host organism 2. Types of Cloning Vectors Vector Host Organism Plasmid Bacteria Small, circular DNA with an antibiotic resistance gene < 5 kb Bacteriophage λ Bacteria Linear DNA that is used for construction of cDNA libraries and genomic libraries < 20 kb Bacterial Artificial Chromosome (BAC) Bacteria Large DNA plasmids used for construction of genomic libraries 100 - 300 kb Yeast Artificial Chromosome (YAC) Yeast Vector that contains centromere, telomeres and ori sequence for replication- used for large clones 50 - 1000 kb General Features Insert Size 3. Plasmid Vectors 1 • Antibiotic resistance gene(s) 2 • Polylinker containing unique restriction endonuclease sites for insertion of DNA fragment in the plasmid 3 • Recombinant plasmid replicated in host bacteria 4. Cloning Using Plasmid Vectors EcoR1 site 1 DNA inserts with EcoRIcompatible ends ampr gene Plasmid vector ampr gene ori DNA ligase Insert EcoRI digestion Plasmid vector ori 2 Transform E. coli with plasmid; Plate on medium + ampicillin amp gene r Amp resistant colonies Recombinant Plasmid ori Isolate colonies for screening Bacterium Chromosome Plasmid (exaggerated in size) Tools of Genetics [10] B. RECOMBINANT DNA: CLONING 15. Cloning Using Bacteriophage Vectors • Ligation of DNA inserts in l phage vector and packaging into phage particles cos cos l phage cloning vector EcoRI digest cos cos l phage DNA ligase 2 • The cos sites at the ends of the l phage vector enables it to circularize in the host bacterium, DNA Inserts thereby preventing integration into the bacterial chromosome. Mix with phage proteins Each recombinant phage forms a plaque Infect E. coli Bacterial lawn Phage plaque C. TYPES OF LIBRARIES 1. Genomic Libraries • DNA is isolated from cells or tissue and digested into millions of fragments comprised of overlapping sequences covering the entire genome. Genomic DNA Restriction endonuclease digestion DNA Fragments • All of the genomic DNA fragments are ligated into a cloning vector such as λ phage and then packaged into particles. Ligate into l phage vector Package into phage particles • Genomic libraries are screened for DNA fragments containing a specific sequence. Phage particles with DNA fragments identified by screening can be amplified in the host organism and the DNA isolated for further sequencing. Genomic library of DNA fragments in λ phage vector Tools of Genetics [11] C. TYPES OF LIBRARIES 2. cDNA Libraries • mRNA is extracted from cells or tissue and used to generate cDNA by reverse transcription. • cDNA libraries contain expressed sequences only, for example, sequences derived from exon of spliced mRNAs such as 5´- and 3´-Untranslated Regions (UTR) and protein coding sequences. • cDNA clones that are sequenced and mapped to the genome are called expressed sequence tags (ESTs) Cells or Tissue RNA extraction mRNA 5´- -AAAAAA-3´ Oligo (dT) Primer complementary to PolyA tail mRNA template -AAAAAA-3´ 3´-T T T T T T-5´ primer 5´- Add Reverse Transcriptase for 1st strand synthesis of DNA using mRNA as template 1st strand 5´3´- -AAAAAA-3´ -T T T T T T-5´ RNase H for degradation of mRNA template; Add primer and DNA polymerase for 2nd strand synthesis 2nd strand 5´3´- -AAAAAA-3´ -T T T T T T-5´ cDNA Ligate into l phage or other cloning vector cDNA Library 3. PCR Libraries • High-throughput sequencing methods such as massively parallel (next-gen) sequencing are capable of analyzing millions of sequences in a library in a single run. • Libraries are constructed from DNA or RNA samples using various procedures that involve DNA fragmentation, amplification, adapter ligation, enrichment and quality control. RNA Genomic DNA Fragmentation Genomic DNA Reverse Transcriptase PCR cDNA Adapter ligation PCR Adapters Sequencing Library Adapter ligation PCR Adapters Sequencing Library Tools of Genetics [12] D. ANALYSIS OF GENE EXPRESSION 1. Microarrays: Gene Expression Profiling • In this technique, mRNA is extracted from a tissue sample, synthesized into cDNA by RT-PCR, and tagged with a fluorescent label. As shown in the example below, cDNA from a reference (normal) sample is labeled with green dye and cDNA from tumor sample with red dye. • The cDNA samples are mixed and hybridized to a microarray that contains DNA fragments (probes) corresponding to thousands of genes (some microarrays contain nearly 50,000 probes). • The signals are scanned by a laser and the red to green fluorescence ratio in each spot calculated. A red signal would indicate that a particular gene is highly expressed in the tumor sample but not in the normal sample, while a green signal would indicate expression of genes in the normal sample, but not in the tumor sample. A yellow signal would indicate about equal levels of gene expression in both samples. Fxn: Gene expression profile Chromosome Contig Sequences mapped to each fragment are spotted on microarray Each spot on the microarray is a known sequence Examples: Gene Sequences, ESTs D. ANALYSIS OF GENE EXPRESSION Tools of Genetics [13] Example: Gene Expression Profiling in Primary Tumors for Breast Cancer 2. RNA-seq RNA-seq is a method that uses massively parallel sequencing technology to sequence a cDNA library that is generated by reverse transcription of either mRNA or total RNA. Millions of cDNA sequence reads are mapped to a reference genome. An incredible amount of information on gene expression is obtained such as relative levels of individual mRNAs, mRNA isoforms, splice variants and expression of novel mRNAs. (A) cDNA library is constructed from either mRNA or total RNA by reverse transcription. (B) Sequencing adaptors are ligated to cDNA fragments for massively parallel sequencing. (C) Sequence reads are mapped to a reference genome and analyzed. Red boxes in C denote exon reads and blue boxes represent splice junction reads.