1. Biochemistry for Nursing Summer semester, 2015 Dr. Mamoun Ahram
Genomics
Biochemistry for Nursing
Summer semester, 2015
Dr. Mamoun Ahram

2. Chromosome vs. chromatin
Chromatin is not condensed and cannot be distinguished from each other before cell division.
Chromosomes: condensed DNA molecules that can be distinguished from other chromosomes at cell division.

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

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

6. 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.

7. 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

8. 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 dividing (known as senescence).
Continuation of shortening telomeres is associated with DNA instability and cell death.

9. 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

10. Coding versus non-coding sequences
The estimated number of genes is 20,000 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.

11. What are the functions of non-coding segments of DNA?
Hypotheses
They are needed to help fold the DNA within the nucleus.
They have played a role in evolution.
The segments are functional but the functions are not yet understood.
I also think that they can protect the DNA from harmful damages of chemical and ionizing radiation.

12. DNA mutations

13. 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.

14. Types of mutations

15. DNA mutations

16. Some Common Hereditary Diseases and Their Causes

17. Polymorphisms
Polymorphisms are also 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.

18. 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.

19. 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.

20. Genomics and biotechnology

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

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

23. Resources http://www.personal.psu.edu/pzb4/electrophoresis.swf

24. 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 +

25. 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

26. 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

28. Restriction sites
Restriction endonucleases recognize specific 4- to 8-bp sequences, called restriction sites, and then cleave both DNA strands at this site

29. Restriction fragments
Restriction endonucleases cut the DNA into fragments called restriction fragments

30. 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)

31. DNA polymorphisms and 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.

32. Example

33. 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

34. Example

35. 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

36. 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

37. Example 1 (continue)

38. Example 2: Paternity testing

39. 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

40. 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

41. 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.

42. 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

43. Method of DNA sequencing
The most common method of DNA sequencing is based on premature termination of DNA synthesis resulting from the inclusion of chain-terminating dideoxynucleotides (which do not contain the deoxyribose 3 hydroxyl group) in DNA polymerase reactions

44. The process…
DNA synthesis is initiated from a primer that allows the DNA polymerase to start working.
Four separate reactions are run, each including deoxynucleotides plus one dideoxynucleotide (either A, C, G, or T)
Incorporation of a dideoxynucleotide stops further DNA synthesis because no 3’- hydroxyl group is available for addition of the next nucleotide

45. Generation of fragments
A series of labeled DNA molecules are generated, each terminating at the base represented by the dideoxynucleotide in each reaction
These fragments of DNA are then separated according to size by gel electrophoresis and detected by exposure of the gel to X-ray film
The size of each fragment is determined by its terminal dideoxynucleotide, so the DNA sequence corresponds to the order of fragments read from the gel

48. Direction of reading the synthesized DNA3’
Direction of reading the synthesized DNA 5’

49. 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

51. 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 nucleotides long. The region between the primers is amplified
all four deoxyribonucleoside triphosphates (dNTPs: dATP, dCTP, dGTP and dTT)
a heat-stable DNA polymerase

52. 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

53. 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

55. PCR cycles
In practice, 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

56. 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

60. 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?