Genomics

1. Genomics Dr. Mamoun Ahram Biochemistry for Nursing First semester 2018-2019

2. Human DNA • In humans, the DNA is made of a sequence of 3 billion bases organized into chromosomes (44 chromosomes called autosomal chromosomes and 2 sex chromosomes-X and Y). • Chromosome 21 is the smallest and chromosome 1 is the largest.

3. Chromosomal regions • There are two types of specialized chromosomal regions: • Centromere • Telomere

4. Centromere • It is a constriction in the middle of chromosomes. • It is responsible for chromosomal movement at cell division. • It divides the chromosome into short and long arms, designated • p (= petite) and q ('g' = grande) • The duplicated chromosomes bound at the centromere are known as sister chromatids. • The centromere contain large repetitive base sequences that do not code for proteins.

5. Telomeres • They are long, noncoding series of a repeating group of nucleotides (TTAGGG) • The tip that seals the ends of chromosomes and protects their structural integrity. • Telomeres also prevent the DNA from bonding to the DNA in other chromosomes or DNA fragments.

6. Telomeres and aging • As we age, telomeres get shorter, chromosomes become less stable, cells dies, and then we die. • A very short telomere is associated with the stage at which a cell stops growing and dividing (known as senescence). • Continuation of shortening telomeres is associated with DNA instability and cell death.

7. Telomerase • Telomerase is the enzyme responsible for adding telomeric sequences to DNA to keep them long. • If telomerase remains active in a cell, the cell would not age and instead would continue to divide. • Think!! cancer

8. Coding versus non-coding sequences • The estimated number of genes is ~22,000 within our DNA. • Coding sequences are genes, which are parts of DNA that are transcribed and translated into proteins. • Non-coding sequences: introns, centromeres, and telomeres • About 2% of all DNA in the human genome actually codes for protein.

9. DNA mutations

10. Mutations in DNA versus mRNA • During transcription, an error that occurs perhaps one out of a million times. That would hardly be noticed in the presence of many correct mRNAs. • If an error occurs during the replication of a DNA molecule, however, the consequences can be far more damaging. • An error in base sequence of DNA is called a mutation. • Some mutations result from spontaneous events. • Others are induced by exposure to a mutagen an external agent like viruses, chemicals, and ionizing radiation.

11. Types of mutations

12. DNA mutations

13. Some Common Hereditary Diseases and Their Causes

14. Polymorphisms • Polymorphisms are variations in the nucleotide sequence of DNA, but they are common within a given population. • Some polymorphisms are responsible for some inherited human diseases. • The location of polymorphisms are linked to other diseases.

15. Single nucleotide polymorphism (SNPs) • They are replacement of one nucleotide by another in the same location along the DNA sequence. • They occur in at least 1% of a specific population and therefore provides a link to a genetic characteristic of that population. • SNPs are the most common source of variations between individual human beings. • SNPs occur throughout the human genome - about one in every 300 nucleotide base pairs. • ~10 million SNPs within the 3-billion-nucleotide human genome.

16. Biological effects of SNPs • The biological effects of SNPs are wide ranging, from being negligible, to normal variations such as those in eye or hair color, to genetic diseases. • Some SNPs can cause a change in the amino acid sequence of a protein, others are “silent”. • They can be linked to a disorder.

17. Genomics and biotechnology

18. What is genomics? • Genomics is the study of whole sets of genes and their functions.

19. Gel electrophoresis • The length and purity ofDNA molecules can be accurately determined by the gel electrophoresis wells - Direction DNA travels - +

20. Resources • http://www.personal.psu.edu/pzb4/electrophoresis.swf • http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html • http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

21. How do DNA segments look like in a gel? • When DNA is stained, they appear as "bands“. • Each band contains thousands to millions of the same DNA molecules or different DNA molecules of the same size. Size Standard Sample 1 Sample 2 - 1000 bp 850 bp 750 bp 600 bp 200 bp 100 bp +

22. Endonucleases • Among the many DNA-binding proteins are endonucleases. • These are enzymes that degrade DNA within the molecule rather than from either end (exonucleases). 5’-Exonuclease 3’-Exonuclease Endonuclease

23. Restriction endonucleases • A class of endonucleases is restriction endonucleases • They are given the name "restriction: because each enzyme recognizes and cuts at a specific sequence • For example, the type II enzyme called EcoRI (isolated from E. coli) cuts DNA only at the hexanucleotide 5'-GAATTC-3‘ • Digestion of DNA with such an enzyme therefore gives the same set of fragments

25. Restriction sites and fragments • Restriction endonucleases recognize specific 4- to 8-bp sequences, called restriction sites, and then cleave both DNA strands at this site. • Restriction endonucleases cut the DNA into fragments called restriction fragments.

26. Advantage of restriction endonucleases • There are many ways by which we can take advantage of restriction endonucleases • One of them is restriction fragment length polymorphism (RFLP). • Because of DNA polymorphisms among individuals, restriction sites can be created or removed. • As a consequence, the pattern of restriction fragment lengths from a region of the genome may differ within and among individuals.

27. Example

28. Restriction fragment length polymorphism • The presence of different fragments in individuals generates a restriction fragment length polymorphism, or RFLP • Remember!! we have two copies of the same DNA (paternal and maternal). Therefore, we should either have inherited the DNA sequence with the same sequence from both or two different DNA sequences

29. Example Homozygous for A Homozygous for B Heterozygous

30. RFLP in the clinic • RFLP can be used as diagnostic tools • For example, if a mutation that results in the development of a disease also causes the generation of distinctive RFLP fragments, then we can tell • if the person is diseased as a result of this mutation • from which parent this allele is inherited

31. Example 1: Disease detection by RFLP(sickle cell anemia) • Note: • in this disease, the person must have both copies of the chromosomes mutated. • If a person has one mutated copy, the person is a carrier. Normal/ carrier Normal Diseases

32. Example 1 (continue)

33. Example 2: Paternity testing

34. Recombinant DNA technology • The basic strategy in molecular cloning is to insert a DNA fragment of interest (e.g., a segment of human DNA) into a carrier DNA molecule (called a vector) • Such vector must be capable of independent replication in a host cell • The result is what is known as a recombinant molecule, that is a new DNA molecule made by joining two different DNA molecules

35. Making of recombinant DNA • Recombinant DNA molecule is made when both DNA fragments (the DNA to be cloned and a vector) are cut by the same restriction endonucleases • When the cut DNA fragments are mixed, they will bind to each other at the cohesive ends

36. Uses of recombinant DNA • Once a recombinant DNA is made, it is inserted into a bacterial cell that synthesize the protein encoded by the inserted gene. • Since bacteria multiply rapidly, there are soon a large number of them, all containing the recombinant DNA and synthesizing the protein encoded by the recombinant DNA. • Examples: insulin, human growth hormone.

37. Polymerase Chain Reaction • Polymerase chain reaction (PCR) is used to amplify specific DNA sequences • The PCR method is extremely sensitive; it can detect a single DNA molecule in a sample

39. Components of PCR reaction • a pair of primers that hybridize to the target DNA. These primers should be specific for the target sequence and which are often about 15-25 nucleotides long. The region between the primers is amplified • all four deoxyribonucleoside triphosphates (dNTPs: dATP, dCTP, dGTP and dTT) • a heat-stable DNA polymerase

40. DNA polymerases • Suitably heat-stable DNA polymerases have been obtained from microorganisms whose natural habitat is hot springs • For example, the widely used Taq DNA polymerase is obtained from a thermophilic bacterium, Thermus aquaticus, and is thermostable up to 94°C

41. PCR cycle • Denaturation, typically at about 93-95°C. At this temperature the hydrogen bonds that hold together the two polynucleotides of the double helix are broken, so the target DNA becomes denatured into single-stranded molecules • Reannealing at temperatures usually from about 50°C to 70°C where the primers anneal to the DNA • DNA synthesis, typically at about 70-75°C, the optimum for Taq polymerase

43. PCR cycles • In practice, 20-30 cycles of reaction are required for effective DNA amplification, with the products of each cycle serving as the DNA templates for the next-hence the term polymerase "chain reaction“ • Every cycle doubles the amount of DNA synthesized in the previous cycle • With each round of DNA synthesis, the newly generated fragments serve as templates in their turn, and within a few cycles the predominant product is a single species of DNA fragment whose length corresponds to the distance between the two original primers • After 30 cycles, there will be over 250 million short products derived from each starting molecule

44. Forensic medicine • An individual DNA profile is highly distinctive because many genetic loci are highly variable within a population • PCR amplification of multiple genes is being used to establish paternity and criminal cases

46. What is DNA sequencing? • DNA sequencing is the process of determining the exact order of the chemical building blocks, that are the A, T, C, and G bases, that make up the genome

47. Molecular fingerprinting • The fact that each person has a molecular profile different from other people is known as molecular (or DNA) fingerprinting Which people have the same exact molecular fingerprint?