1. Tools and technique of molecular biology

2. Polymerase chain reaction (PCR) The polymerase chain reaction is an extremely versatile technique for copying DNA. PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules.

3. PCR Analysis The process follows the principle of DNA replication

5. PRIMER A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. These primers are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases. They are hybredized to a target DNA, which is then copied by the polymerase. minimum primer length used in most applications is 18 nucleotides. Replication starts at the 3'-end of the primer, and copies the opposite strand. In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA.

6. How to see genes...... where in tissues?... where on chromosomes? How to see specific forms of genes? How to clone and amplify genes? southern blot RFLP in situ hybridizationrecombinant DNA genomic libraries

7. How to analyze specific form of genes in genomic DNA? Southern blotting Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. less commonly used due to the capacity of other techniques, such as PCR. Southern blotting are still used for some applications such as measuring transgene copy number in transgenic mice, or in the engineering of gene knockout embryonic stem cell lines.

8. Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection

9. MOLECULAR BIOLOGY – Molecular biology techniques SOUTHERN BLOT combines DNA fragmentation, gel electrophoresis and hybridization to analyze specific DNA sequences

10. Northern blotting The northern blot is used to study the expression patterns of a specific type of RNA molecule as relative comparison among a set of different samples of RNA. RNA is separated based on size and is then transferred to a membrane then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used. Most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. It is used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.

11. Western blotting, or immunoblotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. Flow chart of Western blotting Electrophoresing the protein sample Assembling the Western blot sandwich Transferring proteins from gel to nitrocellulose paper Staining of transferred proteins Blocking nonspecific antibody sites on the nitrocellulose paper Probing electroblotted proteins with primary antibody Washing away nonspecifically bound primary antibody Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine (DAB) precipitate Photographing the immunoblot

12. SDS polyacrylamide-gel electrophoresis (SDS-PAGE)

13. Comparison of Southern, Northern, and Western blotting techniques

14. Hybridization to filter complementary cloning GENOMIC LIBRARY isolate DNA

16. Molecular Diagnosis Nucleic Acid Techniques: Plasmid profiling (when characteristic plasmids are available) RFLP analysis (restriction fragment length polymorphisms - use RE to produce characteristic gel fragments) PCR (amplify key sequences which distinguish target microorganisms) Plasmid Analysis plasmids are small, self- replicating circular DNA molecules found in many bacteria plasmids often code for resistance to antibiotics and certain virulence factors widely-used for tracking resistance in disease outbreaks / pandemics can track transfer of resistance: between hospitals, organisms, and countries weakness - can transfer between microbes Restriction Enzyme Pattern Cut DNA at specific location using natural enzymes: restriction endonucleases get characteristic fragments: “RFLP's / restriction fragment length polymorphisms” see fragments via electrophoresis very accurate – can ID between microbial strains

17. PCR: Polymerase Chain Reaction Making copies of a DNA sequence PCR is conducted in vitro (beaker / test tube) 20 cycles of PCR can allow for a 1,000,000 X amplification of DNA samples Typical PCR reactions use small (ng-mg) quantities of DNA and go through 30-40 amplification cycles PCR has revolutionized R&D in biology / medicine and helped refine criminology and law Uses of Restriction Enzyme Patterns: identification of bacterial populations epidemiology (spread of disease through population), pandemic science study of Tuberculosis in HIV-positive patients combined with DNA fingerprinting (southern blot, etc.) it is a very powerful tool

18. can identify organisms at, or below species level can detect fastidious organisms directly (bacteria, viruses, mycobacteria, fungi and parasites) Commercial kits available: Gen-probe, Microprobe, Digene etc. (all FDA approved) procedures are well- standardized use short, synthetic DNA probes for well understood characteristic sequences Nucleic Acid Probes – MicroArray

19. Gene Therapy Techniques that aim to cure an inherited disease by providing the patient with a correct copy of the defective gene Can include “gene addition” or “gene subtraction”

20. Gene Therapy for Inherited Diseases Germline therapy – uses a fertilized egg, so the gene is present in all cells of the resulting individual Somatic cell therapy - good for inherited blood diseases (e.g., haemophilia, thalassemia; stem cells from the bone marrow) and lung diseases (e.g., cystic fibrosis; periodic inhaling of DNA in rats) no good method available yet for replacing a defective gene (necessary for a dominant one) Current therapies add a gene but can’t replace the defective information yet

21. Gene Therapy and Cancer gene therapy can be applied not only to inherited / infectious diseases but also cancer specific killing of cancer cells using cancer- specific promoters and toxin genes cause tumor cells to synthesize strong antigens that are efficiently recognized by the immune system suitable delivery methods to the cancerous cells are not yet available

22. 4. Stem Cells Stem cells are poised to revolutionize medical science: Re-grow damaged tissues Fix otherwise lethal abnormalities Potential to repair birth defects before symptoms ever appear Cure “incurable diseases” (MS, ALS, etc.)

23. Somatic Stem Cells Somatic stem cells occur in different types: Haematopoietic stem cells – blood-forming stem cells are found in the bone marrow as well as the umbilical cord of newborn babies. Stromal stem cells – (bone marrow cells) can differentiate into cartilage, fat/adipocytes and bone. Neural stem cells –can differentiate into various neural cells including neurons and the myelin-sheath producing oligodendrocytes. Embryonic Stem Cells Derived from cells of the inner cell mass of the blastocyst (<5 day old embryonic cell mass) - typically has less than 160 cells in total. Like Somatics, Embryonic stem cells have two core characteristics: an unlimited capacity to self- replicate the capability (potency) of differentiating into any one of the more than two hundred identified tissue types found in the human body.

24. Molecular markers Molecular marker are based on naturally occurring polymorphism in DNA sequence(i.e. base pair deletion, substitution,addition or patterns). Genetic markers are sequences of DNA which have been traced to specific locations on the chromosomes and associated with particular traits. It can be described as a variation that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like mini satellites.

25. Some commonly used types of genetic markers are RFLP (or Restriction fragment length polymorphism) AFLP (or Amplified fragment length polymorphism) RAPD (or Random amplification of polymorphic DNA) VNTR (or Variable number tandem repeat) Micro satellite polymorphism, SSR (or Simple sequence repeat) SNP (or Single nucleotide polymorphism) STR (or Short tandem repeat) SFP (or Single feature polymorphism) DArT (or Diversity Arrays Technology) RAD markers (or Restriction site associated DNA markers)

26. There are 5 conditions that characterize a suitable molecular marker Must be polymorphic Co-dominant inheritance Randomly and frequently distributed throughout the genome Easy and cheap to detect Reproducible

27. Molecular markers can be used for several different applications including Germplasm characterization, Genetic diagnostics, Characterization of transformants, Study of genome Organization and phylogenic analysis. Paternity testing and the investigation of crimes. Measure the genomic response to selection in livestock

28. RFLP (Restriction fragment length polymorphism) RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs. A RFLP occurs when the length of a detected fragment varies between individuals and can be used in genetic analysis. Advantages: Variant are co dominant Measure variation at the level of DNA sequence, not protein sequence. Disadvantage: Requires relatively large amount of DNA

29. AFLP ( Amplified fragment length polymorphism) In this analysis we can amplify restricted fragments and reduces the complexity of material to be analyzed (approx 1000 folds).it can be used for comparison b/w closely related species only. Advantages: Fast Relatively inexpensive Highly variable Disadvantage: Markers are dominant Presence of a band could mean the individual is either homozygous or heterozygous for the Sequence - can’t tell which?

30. RAPD ( Random amplification of polymorphic DNA) Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. Advantages: Fast Relatively inexpensive Highly variable Disadvantage: Markers are dominant Presence of a band could mean the individual is either homozygous or heterozygous for the Sequence - can’t tell which? Data analysis more complicated

31. Micro satellite polymorphism, SSR or Simple sequence repeat Microsatellites, Simple Sequence Repeats (SSRs), or Short Tandem Repeats (STRs), are repeating sequences of 1-6 base pairs of DNA. Advantages: Highly variable Fast evolving Co dominant Disadvantage: Relatively expensive and time consuming to develop

32. SNP A single-nucleotide polymorphism (SNP, pronounced snip) is a DNA sequence variation occurring when a single nucleotide — A, T,C, or G — in the genome (or other shared sequence) differs between members of a species or paired chromosomes in an individual. Used in biomedical research,crop and livestock breeding programs.

33. STR A short tandem repeat (STR) in DNA occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The pattern can range in length from 2 to 16 base pairs (bp) (for example (CATG) n in a genomic region) and is typically in the non-coding intron region Used in forensic cases. used for the genetic fingerprinting of individuals