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

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

3. 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

4. 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)

5. 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.

7. 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.

8. 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.

9. 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 bp, or around 1m.
Mitochondria: bp.
Chloroplast: 1.5x 105 bp or µm
Human Genome:
Nucleus: 3x 1069bp or 1m.
Mitochondria: 16 to 20 x103 bp.

10. 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?

11. 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%?

12. 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

13. 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??

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

15. 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

16. 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

17. 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.

18. 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.  

19. 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.

20. 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

21. Basic molecular biology techniques
Isolating nucleic acids

22. Basic molecular biology techniques
Isolating nucleic acids
Cutting DNA into fragments

23. DNA can be reproducibly split into fragments by restriction endonucleases

24. 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.

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

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

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

28. 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

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

30. The polymerase chain reaction (PCR)

31. DNA polymerases
dATP
dTTP
dGTP
dCTP

32. 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

33. 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

34. 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.

35. Transcriptome analysis using microarrays

36. Constitutive expression of a genes transcribed at a constant level

37. 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.

38. 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

39. Genomic sequence

40. Finding open reading frames

41. Finding open reading frames
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Expasy server

42. Sequence from the E. coli genome

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

44. 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

45. 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)

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

47. 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

48. 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 Genomes 3 (© Garland Science 2007)

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

50. Whole genome studies
Tiling assays

51. Working with proteins Separating proteins
Analyzing proteins and their interactions

52. Separating proteins on polyacrylamide gels

53. Immunoblot (Western blot)

54. Proteins can be sequenced

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

56. Liquid chromatography is used to separate
peptides before mass spectrometry

57. 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 Genomes 3 (© Garland Science 2007)

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