Genomics

Biochemistry for NursingSummer semester, 2015

Dr. Mamoun Ahram

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Chromosome vs. chromatin

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• 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.

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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.

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Chromosomal regions

• There are two types of specialized chromosomal regions:– Centromere– Telomere

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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.

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

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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.

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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

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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.

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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.

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DNA MUTATIONS

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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.

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Types of mutations

DNA mutations

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Some Common Hereditary Diseases and Their Causes

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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.

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

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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.

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GENOMICS AND BIOTECHNOLOGY

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What is genomics?

• Genomics is the study of whole sets of genes and their functions.

Gel electrophoresis

• The length and purity of DNA molecules can be accurately determined by the gel electrophoresis

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Direction DNA travels

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

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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 St

andard

1000 bp850 bp750 bp600 bp

200 bp100 bp

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Sam

ple 1

Sam

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Endonucleases

• Among the many DNA-binding proteins are endonucleases.

• These are enzymes that degrade DNA within the molecule rather than from either end (exonucleases).

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5’-Exonuclease

3’-Exonuclease

Endonuclease

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

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Restriction sites

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

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Restriction fragments

• Restriction endonucleases cut the DNA into fragments called restriction fragments

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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)

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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.

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Example

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

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Example

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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

Example 1: Disease detection by RFLP(sickle cell anemia)

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NormalNormal/carrierDiseases

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.

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Example 1 (continue)

Example 2: Paternity testing

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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

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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

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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.

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

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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

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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

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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

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485’

3’

Direction of reading the synthesized

DNA

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

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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

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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

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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

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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

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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

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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?

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