Chapter 2 - Fundamental Technologies Eukaryotic gene organization Molecular cloning Genomic and cDNA libraries Polymerase chain reaction (PCR) Chemical synthesis of DNA DNA sequencing technologies Sequencing whole genomes Genomics (and other –omics) Genome engineering using CRISPR Technology

Eukaryotic gene organization Enhancers Silencers Insulators

RNA Processing in Eukaryotes FIGURE 5-15 Overview of RNA processing. RNA processing produces functional mRNA in eukaryotes. Transcription of eukaryotic protein-coding genes: RNA polymerase starts transcription at gene nucleotide +1, which is upstream of the codon that encodes the first amino acid. RNA polymerase stops transcription downstream of the translation STOP codon. The 5’ and 3’ untranslated regions (UTRs) are retained in the fully processed mRNA. 5′ cap: added during formation of the primary RNA transcript. Poly A tail: primary transcript is cleaved at a specific site downstream of the translation STOP codon and multiple (100–200) A residues (not encoded by the gene DNA template) are added enzymatically by poly (A) polymerase poly(A) tail stabilizes mRNAs in the nucleus and cytoplasm and is involved in mRNA translation Splicing: removes introns and joins exons The β-globin gene contains three protein-coding exons (constituting the 147–amino acid coding region) and two intervening noncoding introns, which interrupt the protein-coding sequence between the codons for amino acids 31 and 32 and 105 and 106, and are spliced out of the fully processed mRNA.

Structure of the 5′ methylated cap FIGURE 5-14 Structure of the 5′ methylated cap. Eukaryotic precursor mRNAs are processed at both 5’ and 3’ ends to form functional mRNAs. 5′ methylated cap added by enzymes as 5’ end emerges from RNA polymerase protects mRNA from enzymatic degradation, assists export to the cytoplasm, and is bound by a protein factor required to begin translation by a ribosome in the cytoplasm. 5′→5′ linkage of 7-methylguanylate to the initial nucleotide of the mRNA molecule and methyl group addition to the 2′ hydroxyl of the ribose of base 1 occur in all animal and higher plant cells yeasts lack the methyl group on base 1 vertebrate cells also methylate the base 2 ribose

Basic Transcriptional Mechanisms Life Cycle of mRNA (https://www.youtube.com/watch?v=h7FHqaetMS0) mRNA splicing (https://www.youtube.com/watch?v=FVuAwBGw_pQ)

Eukaryotic gene expression

Insulators Two kinds of insulator functions. (A) Some insulators may function as barriers against the encroachment of adjacent genomic condensed chromatin. (B) Some insulators may serve as positional enhancer-blocking elements that prevent enhancer action when placed between enhancer and promoter, but not otherwise.

Molecular Cloning: Recombinant DNA cloning procedure Requires a DNA fragment and a cloning vector Plasmid Cloning (https://www.youtube.com/watch?v=2vrt87wjOBg)

Table 2.1

Figure 2.1 Eco RI cuts DNA to produce complementary (sticky) ends

Restriction enzymes & DNA methylation

Figure 2.1 (Continued)

Mapping of restriction enzyme sites

Cloning vectors and their insert capacities Vector system Host cell Insert capacity (kb) Plasmid E. coli 0.1-10 Bacteriophage l 10-20 Cosmid 35-45 Bacteriophage P1 80-100 BAC (bacterial artificial chromosome) 50-300 P1 bacteriophage-derived AC 100-300 YAC Yeast 100-2,000 Human AC Cultured human cells >2,000

Figure 2.3 Some other important enzymes used to prepare DNA for cloning Alkaline phosphatase-removes 5’ phosphate (P) groups of DNA molecules

Figure 2.4 Ligation of two different DNA fragments after digestion with BamHI using T4 DNA ligase T4 DNA ligase –joins 5’ phosphate (P) groups of DNA molecules to 3’ hydroxyl (OH) groups of DNA

Figure 2.5 Plasmid cloning vector pUC19 Three important vector features Cloning site (MCS) Origin of replication (Ori) A selectable marker (Ampr) Figure 2.5 Plasmid cloning vector pUC19

Figure 2.6 Cloning target DNA into pUC19

pUC18/19 pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp in length. They are identical except that they contain multiple cloning sites (MCS) arranged in opposite orientations. pUC18/19 plasmids contain: (1) the pMB1 replicon rep responsible for the replication of plasmid (source – plasmid pBR322). The high copy number of pUC plasmids is a result of the lack of the rop gene and a single point mutation in rep of pMB1; (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – plasmid pBR322); (3) region of E.coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5’-terminal part of the lacZ gene encoding the N-terminal fragment of beta-galactosidase (source – M13mp18/19). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa) complementation with a defective form of beta-galactosidase encoded by host (mutation lacZDM15). In the presence of IPTG, bacteria synthesize both fragments of the enzyme and form blue colonies on media with X-Gal. Insertion of DNA into the MCS located within the lacZ gene (codons 6-7 of lacZ are replaced by MCS) inactivates the N-terminal fragment of beta-galactosidase and abolishes alfa-complementation. Bacteria carrying recombinant plasmids therefore give rise to white colonies.

Figure 2.7 Electroporation can be used for DNA transformation of cells

Figure 2.8 Blue/white screening of bacterial cells to identify host cells transformed with pUC19 carrying the cloned target DNA

Recombinational Cloning Recombinational Cloning. Invitrogen’s Gateway® technology facilitates cloning of genes, into and out of, multiple vectors via site-specific recombination. Once a gene is cloned into an Entry clone you can then move the DNA fragment into one or more destination vectors simultaneously.

Some antibiotics commonly used as selective agents Description Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by b-lactamase, which cleaves the b-lactam ring of amp Hygromycin B (HygB) Blocks translocation from amino acyl site to peptidyl site Kanamycin (Kan) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase Neomycin (Neo) Streptomycin (Str) Blocks protein initiation complex formation and causes misreading during translation Tetracycline (Tet) Binds to 30S ribosomal subunit and inhibits protein synthesis; tetr gene encodes a protein which prevents transport of tet into the cell

Library Construction and Screening Genomic (gene) libraries cDNA libraries Library screening

Eukaryotic gene organization Enhancers Silencers Insulators

Genomic library construction

Screening a genomic library using DNA hybridization to a (radio-)labeled DNA probe Note: a cDNA is commonly (radio-)labeled and used as a DNA probe to screen a genomic library

Production of a (radio-)labeled DNA probe by the random primer method [uses the Klenow fragment of DNA polymerase] 5’ 3’ Denature in presence of nonamer primers 5’ 3’ 3’ 5’

The first step in making a cDNA library: Purification of polyadenylated mRNA using oligo(dT)-cellulose Note: selection of the proper source (organ, tissue) of the RNA is critical here!

Complementary DNA or cDNA cloning: cDNA library construction Note: ds cDNAs are typically placed in a cloning vector such as bacteriophage lambda (l) or a plasmid

There are several possible ways to screen a cDNA library Using a DNA probe with a homologous sequence (e.g., a homologous cDNA or gene clone from a related species) Using an oligonucleotide probe based on a known amino acid sequence (requires purification of the protein and some peptide sequencing) Using an antibody against the protein of interest (note: this requires use of an expression vector)

Screening a cDNA library using DNA hybridization to a (radio-)labeled DNA probe

Screening a cDNA library with a labeled oligonucleotide probe based on a known peptide sequence

Using polynucleotide kinase and g-32P-labeled ATP to radiolabel oligonucleotide probes

Immunological screening of an expression cDNA library with a primary antibody and labeled secondary antibody; note the label is often an enzyme label like alkaline phosphatase or horseradish peroxidase, but it can also be 125I

The Polymerase Chain Reaction (PCR) 72° 72° See https://www.youtube.com/watch?v=JRAA4C2OPwg PCR is a cloning method without a host Thermus aquaticus, a hot spring bacterium, produces Taq polymerase Taq polymerase, unlike E. coli DNA polymerase, is not denatured at 95°C Kary Mullins invented PCR and won a Nobel Prize for his work in 1993 72° 72°

Figure 2.21 Addition of restriction enzyme recognition sites to primer sequences to facilitate cloning

Figure 2.22 Cloning of PCR products without using restriction enzymes

Figure 2.32 Chemical and enzymatic synthesis of a gene Chemical synthesis of DNA: Oligonucleotide synthesis using phosphoramidite chemistry can be used to make primers, genes, and even entire genomes (read the Box on Synthetic Genomes on p. 43).

DNA Sequencing Technologies Sanger sequencing (Dideoxy Sequencing) Pyrosequencing Sequencing using reversible chain terminators (i.e., sequencing by synthesis) Sequencing by single molecule synthesis See https://www.youtube.com/watch?v=jFCD8Q6qSTM for explanations of Sanger sequencing, sequencing by synthesis, sequencing by ligation, and ion semiconductor sequencing See also https://www.youtube.com/watch?v=Wq35ZXyayuU for Nanopore sequencing with the MinION device- very cool!

Characterization: DNA Sequencing The dideoxynucleotide structure (note 3’H) is the key to dideoxy DNA sequencing

Figure 2.34 Incorporation of a dideoxynucleotide terminates DNA synthesis

Figure 2.35 Dideoxynucleotide/Sanger method for DNA sequencing

Automated DNA sequencing Capillary electrophoresis

Figure 2.36 Pyrosequencing is based on the detection of pyrophosphate released during DNA synthesis

Figure 2.37 Sequencing using reversible chain terminators

Figure 2.38 Real-time single-molecule sequencing [with one molecule of DNA polymerase (orange shape) attached to a nanoscale well]

Figure 2.41 Generation of clusters of sequencing templates; denatured genomic DNA library fragments are captured on a glass slide for Next-Gen Sequencing

Figure 2.42 Genomic sequence assembly merges all of the sequence data

Figure 2.44 Genome annotation looks for conserved sequence features and protein coding regions (open reading frames) in prokaryotes (A) and eukaryotes (B)

Figure 2.45 Transcriptomics or gene expression profiling (which looks at RNA transcripts) on a whole genome basis can be done using a DNA microarray See https://www.youtube.com/watch?v=VNsThMNjKhM Courtesy of N. Anderson, University of Arizona.

DNA microarray analysis can reveal differences in gene expression in fibroblasts under different experimental conditions.

Figure 2.48 Transcriptomics or gene expression profiling (which looks at RNA transcripts) on a whole genome basis can also be done using a High-throughput RNA sequencing (RNA Seq) Adapted with permission from Wang et al., Nat Rev Genet. 10:57–63, 2009.

Figure 2.49 Proteomics can involve 2D PAGE

Figure 2.50 Proteomics can involve peptide mass fingerprinting

Proteomics can be done by amino acid sequencing of peptides using a mass spectrometer and matching the data obtained to a gene/protein database.

Figure 2.52 Proteomics can be done by protein expression profiling with an antibody microarray See https://www.youtube.com/watch?v=-8AUIHyXKG4

Figure 2.56 The yeast two-hybrid assay can be used to detect protein-protein interactions See https://www.youtube.com/watch?v=QpZ5Cv7YVvk

Figure 2.58 Tandem affinity purification to detect multiprotein complexes

Figure 2.59 Metabolomics involves the comprehensive examination of small molecules

Figure 2. 17 Genome editing with CRISPR-Cas9 See https://www. youtube Figure 2.17 Genome editing with CRISPR-Cas9 See https://www.youtube.com/watch?v=2pp17E4E-O8 and https://www.youtube.com/watch?v=jAhjPd4uNFY Adapted by permission from Macmillan Publishers Ltd. from Yosef and Qimron, Nature 519:166–167, 2015.

Figure 2.17 Adapted by permission from Macmillan Publishers Ltd. from Yosef and Qimron, Nature 519:166–167, 2015.

Figure 2.18

Figure 2.19