Techniques in Molecular Biology 2017 Fall Lecture -1- by Jasmin sutkovic 5 .10.2017 12.10.2017

Schedule Time Lecture: Thursday 13:45- 15:30 RDC classroom Lab: Friday 9:30-12:00 RDC (Research and Development Center)

Let`s Lab! LAB EXPERIMENT 1: Introduction to Molecular Biology laboratory techniques and biosafety LAB EXPERIMENT 2: Protein Quantification via Bradford methods LAB EXPERIMENT 3: DNA isolation (genomic) from plant tissue LAB EXPERIMENT 4: DNA isolation from saliva LAB EXPERIMENT 5: DNA quantification LAB EXPERIMENT 6: Gel electrophoresis LAB EXPERIMENT 7: Making Competent Cells

LAB EXPERIMENT 8: Restriction enzyme digest LAB EXPERIMENT 9: Plant Protein Isolation- Rapid isolation of protein for SDS-PAGE analysis LAB EXPERIMENT 10: PCR (Polymerase chain reaction) LAB EXPERIMENT 11: Real-time PCR (real-time Polymerase chain reaction)

Techniques Characterize, isolate, and manipulate the molecular components of cells and organisms. These components include  DNA, the repository of genetic information;  RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus, the ribosome; Proteins, the major structural and enzymatic type of molecule in cells.

Genome The genome is all the DNA in a cell. All the DNA on all the chromosomes Includes genes, intergenic sequences, repeats Specifically, it is all the DNA in an organelle. Eukaryotes can have 2-3 genomes Nuclear genome Mitochondrial genome Plastid genome If not specified, “genome” usually refers to the nuclear genome.

Some Parameters Quantity of DNA per nucleus: about one picogram (1pg=10-12) Total Length of DNA: expressed in base pairs (pb), kilobases (kbp), megabases (1Mb=106 bp) Diameter of DNA: 20 A. A DNA stretch of 1 A length has a mass of 193/6.1023g. One base pair has a mass of 660/N, or 660/6.1023g.

Size of different genome Virus (DNA or RNA): from 103 to 105 bp, or 1 to 10 µm enrolled. Bacteria: 106 or 107 bp, or around 1 mm. Yeast: 1.2x 107 bp. Higher Plants: Nucleus: 108 to 1010 bp, or around 1m. Mitochondria: 1.8105 bp. Chloroplast: 1.5x 105 bp or µm Human Genome: Nucleus: 3x 1069bp or 1m. Mitochondria: 16 to 20 x103 bp.

Human Genome 22 autosome pairs + 2 sex chromosomes 3 billion base pairs in the haploid genome Where and what are the 30,000 to 40,000 genes? Is there anything else interesting/important?

Human genome has 3.2 billion base pairs of DNA About 3% codes for proteins About 40-50% is repetitive, made by (retro)transposition What is the function of the remaining 50%?

Genomics, Genetics and Biochemistry Genetics: study of inherited phenotypes Genomics: study of genomes Biochemistry: study of the chemistry of living organisms and/or cells Revolution lauched by full genome sequencing Many biological problems now have finite (albeit complex) solutions. New era will see an even greater interaction among these three disciplines

One of the main objective of molecular biology and genetics: Finding the function of the gene Genes were originally defined in terms of phenotpyes of mutants. Now we have sequence of lots of DNA from a variety of organisms, so.. Which portion of DNA actually do something. What do they do? Code for protein or some other product? Used in replication, etc??

Genome Structure Distinct components of genomes Abundance and complexity of mRNA Normalized cDNA libraries and ESTs Genome sequences: gene numbers Comparative genomics

Much DNA in large genomes is non-coding Complex genomes have roughly 10x to 30x more DNA than is required to encode all the RNAs or proteins in the organism. Contributors to the non-coding DNA include: Introns in genes Regulatory elements of genes Multiple copies of genes, including pseudogenes Intergenic sequences Interspersed repeats

Basics of Biosafety Principles and practices employed Working Safely with Biological Materials Principles and practices employed to protect laboratory personnel and the environment from exposure or infection while working with living organisms, biological materials, or agents. Included any materials that may be potentially infectious. Includes recombinant DNA research

Some Lab Rules Laboratory exercises should be read before the laboratory period and work should be planned.   Place bags, lab coats, books etc. in specified locations _NEVER ON THE BENCH TOPS No eating or drinking in the laboratory. Do not store food in the laboratory. No pipetting by mouth. Use mechanical pipetting devices only. Wear laboratory coats, disposable gloves, and safety glasses when appropriate.

Keep all noxious and volatile compounds in the fume hood Keep all noxious and volatile compounds in the fume hood. Do not touch broken glassware with your hands. Dispose off broken glass in appropriate receptacles. Do not toss out into regular trash. Live cultures can be treated with Clorox bleach or autoclaved. Do not toss out into regular trash or down drains without autoclaving.  Do not use plastic or polycarbonate containers, test tubes, pipettes etc. with phenol and or chloroform. Instead use polypropylene or glass with these organic compounds. Make sure to use gloves, goggles and lab coats when handling these chemicals.  

Know the potential hazards of the materials, facilities, and equipment with which you will work. Know the location and proper use of fire extinguishers, eyewash stations, and safety showers. Do not dispose of hazardous or noxious chemicals in laboratory sinks. Use proper containers in fume hood. Report all accidents to the instructor immediately.

Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments Hybridization techniques Genomics Sequencing genomes Analyzing genome sequences Proteomics Separating proteins Analyzing proteins

Basic molecular biology techniques Isolating nucleic acids

Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments

DNA can be reproducibly split into fragments by restriction endonucleases

DNA fragments can be separated by size in agarose or polyacrylamide gels Because of the phosphates in the sugar phosphate backbone, nucleic acids are negatively charged. In an electric field nucleic acids will move towards the positive pole. Smaller fragments move faster than larger fragments through the pores of a gel. Very large DNA molecules are separated from each other by special types of electrophoresis, e.g. pulsed field electrophoresis.

Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments

Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments DNA can be amplified by Cloning PCR

DNA cloning and construction of DNA libraries Cloning in a plasmid vector Genomic library cDNA library

Vectors for DNA cloning A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes

Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments DNA can be amplified by Cloning PCR

The polymerase chain reaction (PCR)

DNA polymerases dATP dTTP dGTP dCTP

Basic molecular biology techniques Isolating nucleic acids Cutting DNA into fragments Ligating DNA fragments Amplifying DNA fragments Hybridization techniques In molecular biology, hybridization (or hybridisation) is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA

DNA fragments separated by gel electrophoresis Hybridization probes can be used to detect the presence of their complementary sequence in a number of hybridization applications (Table 1). Table 1. Hybridization methods. Method Target Southern blot DNA fragments separated by gel electrophoresis Northern blot RNA fragments separated by gel electrophoresis Slot/dot blot Total DNA or RNA Colony blot DNA or RNA in microbial colonies Fluorescent in situ hybridization (FISH) DNA or RNA in microbial cells Microarray DNA which is hybridized to probes on an array Quantitative PCR DNA fragments during PCR amplification

Single-stranded nucleic acids can bind to each other by base pairing if they contain complementary sequences Using a single-stranded labeled probe complementary base pairing is able to detect specific nucleic acids among many different nucleic acids. If the probe is used to detect DNA, the analysis is called DNA blot (Southern) analysis. If an RNA fragment is detected, the analysis is called RNA blot (northern) analysis.

Transcriptome analysis using microarrays https://www.youtube.com/watch?v=VNsThMNjKhM

Constitutive expression of a genes transcribed at a constant level

Genomics Sequencing of genomes Split genome into pieces and sequence all pieces. Assembling the sequence (computer). Sequence analysis (annotation 1) Identify genes and other elements in sequence. Functional analysis (annotation 2) Determine function of identified elements.

How to find genes in a genome sequence Protein-coding genes Find open reading frames (protein-coding sequences) Find sequence with a codon bias Find upstream regulatory sequences (e.g. CpG islands) Find exon-intron boundaries Genes coding for functional RNAs Find consensus sequences for tRNAs and ribosomal RNAs Find specific RNA secondary structures (e.g. stem loops) Find upstream regulatory sequences

Genomic sequence

Finding open reading frames

Finding open reading frames gagtccagttgaaaagcaactggaatccccttatagataaattaatatctattttaaaattgaatagtttttattctagtttcgttttaagattaataaaattatgtctaaccaagtatttactactttacgcgcagcaacattagctgttattttaggtatggctggtggcttagcagtaagtccagctcaagcttaccctgtatttgcacaacaaaactacgctaacccacgtgaggctaatggtcgtattgtatgtgcaaactgtcacttagcgcaaaaagcagttgaaatcgaagtaccacaagctgttttacctgatactgtttttgaagctgttattgaacttccatacgataaacaagttaaacaagttttagctaatggtaaaaaaggtgacttaaacgttggtatggttttaattttaccagaaggttttgaattagcaccaccagatcgcgttccggcagaaattaaagaaaaagttggtaacctttactaccaaccatacagtccagaacaaaaaaatattttagttgttggtccagttccaggtaaaaaatacagtgaaatggtagtacctattttatctccagatcctgctaaaaataaaaacgtttcttacttaaaatatcctatttattttggtggtaatcgtggtcgtggtcaagtatatccagatggtaaaaaatcaaacaacactatttacaacgcatcagcagctggtaaaattgtagcaatcacagctctttctgagaaaaaaggtggttttgaagtttcaattgaaaaagcaaacggtgaagttgttgtagacaaaatcccagcaggtcctgatttaattgttaaagaaggtcaaactgtacaagcagatcaaccattaacaaacaaccctaacgttggtggtttcggtcaggctgaaactgaaattgtattacaaaaccctgctcgtattcaaggtttattagtattcttcagttttgttttacttactcaagttttattagttcttaagaaaaaacaattcgaaaaagttcaattagcagaaatgaacttctaatatttaattttttgtagggctgctgtgcagctcctacaaattttagtatgttatttttaaagtttgatatactgaaaacaaagttctacttgaacgatatttagcttttaatgcTATAATATagcggactaagccgttggcaatttagctgccaattaattttattcgaaggatgtaaacctgctaacgatatttatatataagcattttaatactccgagggaggcctctaacctttagcaagtaagtaaacttccccttcggggcagcaaggcagcagatttaaattctccaaaggaggcagttgatatcagtaaaccccttcgatgactctggcattgatgcaaagcatggggaaactaaagttcctccactgcctccttccccttccctttcgggacgtccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtccccttacgggacgtcagtggcagttgcgaagtattaatattgtatataaatatagaatgtttacatactccgaaggaggacgtcagtggcagtggtaccgccactgctattttaatactccgaaggagcagtggtggtcccactgccactaaaatttatttgcccgaagacgtcctgccaactgccgaggcaaatgaattttagtggacgtcccttacgggacgtcagtggcagttgcctgccaactgcctccttccccttcgggcaagtaaacttgggagtattaacataggcagtggcggtaccacaataaattaatttgtcctccttccccttcgggcaagtaaacttaggagtatgtaaacattctatatttatatactcccatgctttgccccttaagggacaataaataaatttgtccccttcgggcaaataaatcttagtggcagttgcaaaatattaatatcgtatataaatttggagtatataaataaatttggagtatataaatataggatgttaatactgcggagcagcagtggtggtaccactgccactaaaatttatttgcccgaaggggacgtcctgccaactgccgatatttatatattccctaagtttacttgccccatatttatatattcctaagtttacttgccccatatttatattaggacgtccccttcgggt Expasy server

Sequence from the E. coli genome

The E. coli genome High gene density on both strands of the E. coli genome.

Genes = all DNA sequences that are transcribed into RNA Protein-coding genes 5’ UTR coding region = open reading frames 3’ UTR 5’ - - 3’ Translation start Translation stop protein-coding gene = DNA transcribed into mRNA UTR = untranslated region

Exons and introns in eukaryotic genes 5’ UTR 3’ UTR Features that can be used to find genes in eukaryotic sequences: Codon bias. Exon-intron boundaries. Upstream sequences: in vertebrates CpG islands (in 40-50% of human genes). Figure 5.4 Genomes 3 (© Garland Science 2007)

Verifying the identity of a gene Homology search Experimental techniques Northern hybridization Zoo-blotting

Verifying the identity of a gene Homology search BLAST MSNQVFTTLR AATLAVILGM AGGLAVSPAQ AYPVFAQQNY ANPREANGRI VCANCHLAQK AVEIEVPQAV LPDTVFEAVI ELPYDKQVKQ VLANGKKGDL NVGMVLILPE GFELAPPDRV PAEIKEKVGN LYYQPYSPEQ KNILVVGPVP GKKYSEMVVP ILSPDPAKNK NVSYLKYPIY FGGNRGRGQV YPDGKKSNNT IYNASAAGKI VAITALSEKK GGFEVSIEKA NGEVVVDKIP AGPDLIVKEG QTVQADQPLT NNPNVGGFGQ AETEIVLQNP ARIQGLLVFF SFVLLTQVLL VLKKKQFEKV QLAEMNF BLAST = Basic Local Alignment Search Tool

Case study, yeast genome 6274 ORFs Orphans= genes of unknown function; single orphans = unique genes not found in databases. Additional methods for identifying the function of genes: Comparative genomics. cDNA sequencing. Transposon tagging. Figure 5.28 Genomes 3 (© Garland Science 2007)

Finding the function of a gene (product) Computer based analysis Homology search Experimental analysis Gene inactivation Overexpression

Whole genome studies Tiling assays

Working with proteins Separating proteins Analyzing proteins and their interactions

Separating proteins on polyacrylamide gels

Immunoblot (Western blot)

Proteins can be sequenced

Complex mixtures of proteins can be analyzed by mass spectrometry MALDI-TOF = Matrix-assisted laser desorption ionization – time of flight

Liquid chromatography is used to separate peptides before mass spectrometry

Mouse liver proteins Mass Spectrometry Example of spots on a gel obtained after 2D electrophoresis. Spots of interest are cut out and protein identified by mass spectrometry. Figure 6.11 Genomes 3 (© Garland Science 2007)

Protein interaction map of yeast Each dot is a protein. Red dot: essential protein. Figure 6.20a Genomes 3 (© Garland Science 2007)