Chapter 15 Molecular Genetics Genome Projects

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Chapter 15- Molecular GeneticsGenome Projects

• By the late 1980s, several Genome Projects had
  begun, involving international collaboration.
• Among the goals
• - To improve plant and animal products for human
  use
• - to attain better treatments for genetic
  diseases
• - To better understand the phylogenetic
  relationships among living organisms.
• Mapping genes in chromosomes
• P ? the short arm of a chromosome (from
  centromere to end)
• Q ? the long arm.
• Regions within chromosomes are referred to as
  p-, where the is a specific location of a
  gene within the p arm of a certain chromosome.

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The Human Genome Project

• Overall goal
• - To determine the 3 billion sequence of DNA
  bases that make up the 23 pairs of human
  chromosomes (22 pairs of autosomes, and X / Y sex
  chromosomes)
• - To map genes to specific chromosomes and
  regions within them.
• - A gene is a region within a DNA molecule
  responsible for encoding a polypeptide. Proteins
  are made of polypeptides, linear molecules made
  of aminoacids.
• General methods
• - Using simpler organisms first. The DNA of
  these organisms uses a similar code (same
  nitrogenous bases A, T, C, G), but contain much
  smaller numbers of bases. Thus, their genomes are
  smaller.
• - Later, some of these simpler organisms became
  very useful, since they have short life cycles
  (reproduce quickly and have short lives), and are
  easy to culture , thus allowing scientists to use
  them as test organisms.
• - Among the first prokaryotes sequenced were
  virus, and bacteria. Much knowledge has been
  gained from Escherichia coli, a bacteria that
  lives in animals digestive tracts.
• - The first eukaryotes sequenced were types of
  yeast, Saccharomyces, an unicellular organism.
• - The first multicellular (eukaryote) sequenced
  was the nematode worm, Caoenorhabdites.

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Development of Caenorhabditis (nematode worm

• The fate of each cell during development has been
  mapped out for this worm, from zygote to adult.
• The embryonic origin of each cell that composes
  the worm (959 cells) has been identified, as well
  as the timing of appearance of new cells as
  result of mitotic cell divisions.
• For this worm, it is known which genes control
  the development of which types of cells.

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Functional genomics The study ofDNA
sequenceinformation
A)
B)
C)

• ? Which specific bases are included in a
  gene
• ? The order in which base pairs appear
• ? The functional meaning of segments of the
  sequence (what aminoacids are encoded by each
  segment)
• - After the sequence of a gene has been
  determined
• 1) A computer examines the triplets of base
  pairs and it determines the corresponding linear
  sequence of aminoacids. For this, it has to
  eliminate all introns (non-coding segments)
• 2) Additional computer analysis predicts the
  tri-dimensional structure of the protein
  (tertiary structure)
• 3) Normal and altered (mutant) protein structure
  can be compared. Changes that may correspond to
  genetically determined diseases may be
  identified.

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More Functional Genomics
Mutant genes may cause differences in the
binding of enzymes and their substrata (enzyme-su
bstratum complex). These changes may result in
reduced ability, or lack of ability to carry
out certain enzymatic processes.

• Many genes code for specific enzymes. Variations
  in these genes may cause differences among
  individuals in performing the reaction catalyzed
  by certain enzymes.
• The cytochrome p-450 gene encodes for the enzyme
  Cytochrome p-450, required for breaking down
  certain toxins and medicines. Computer models
  help predict the ability of different individuals
  (with variations in the cytochrome p-450 gene) to
  carry out these reactions.
• Most genes interact with other genes, and the
  expression of the effect of one is affected by
  the expression of others. Many diseases also
  involve numerous genes. Microarray analysis is a
  technique that allows examining the effect of
  many genes at once, and to identify genes that
  may be associated specific diseases.

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Techniques in genetic engineering

• 1) Using Restriction Enzymes (Restriction
  Endonucleases) (REs)
• - REs cut double-stranded DNA by recognizing
  and cleaving at specific nucleotide sequences,
  ranging from 4 to 10 base pairs (bp)
• - These enzymes were originally isolated from
  bacterial cells, but they will cut any sample of
  DNA, where the sequence they recognize is found.
• - About 200 different REs are known and many
  are used routinely for different purposes. They
  differ in the specific sequence of nucleotides
  they recognize, and thus, the places at which
  they cut DNA.
• - Using REs is a general technique used for
  many purposes, ranging from sequencing a DNA
  sample, to making copies of a DNA segment, and
  more.
• 2) Making Recombinant DNA DNA that has segments
  from various sources (usually different types of
  organisms).
• - This technique is generally used for one of
  two main purposes
• ? Make many copies of a sequence of DNA of
  interest, by inserting into a culture of an
  organism that will replicate it (clone it)
• ? To obtain large quantities of a protein of
  interest (an enzyme or hormone).
• - Several requirements in Recombinant DNA
• a- identifying a gene and separating it from a
  chromosome (using REs)
• b- a vector carrier molecule of DNA into which
  the gene can be inserted. Bacterial plasmids are
  most commonly used.
• c- A method of joining the gene into the vector
  molecule (enzyme DNA ligase is used)
• d- The vector molecule must be introduced into a
  host organism that is going to recognize it as
  its own and make many copies of it for our
  purposes.
• e- A method of recognizing cells that have
  replicated the recombinant DNA.

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Techniques Recombinant DNA

• Goal Making large quantities of Human Growth
  Hormone (GH)
• Segment of human DNA known to contain the gene
  for GH is cut from chromosome
• Bacterial plasmid is cut and human segment
  inserted to make a new plasmid containing the
  human gene.
• New plasmid (Recombinant DNA) is added to live
  bacteria culture. Bacteria take up the new
  plasmid.
• Bacteria replicate (clone) the new plasmid as its
  own, making many copies.
• Bacteria make the human GH of interest, in large
  numbers.

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Techniques3) DNA sequencing

• - Based on the elongation of DNA chains by DNA
  polymerase.
• - Involves breaking an existing DNA sequence
  (that one wants to discern), and allowing it to
  rearrange again using marked elements. It
  requires
• 1) sample of DNA from organism of interest cut
  into pieces (using restriction enzymes), and
  separated in single strands. Four separate
  batches are used, one for each base (A,C,G,T)
• 2) modified nucleotides (can be attached on one
  side but not the other). Will result in terminal
  sites of DNA chains.
• 3) Primers Short sections of DNA that act as
  models from which synthesis is initiated.
• 4) nucleotides so they can be added to the
  growing chains
• 5) DNA polymerase (enzyme that elongates chains
  of DNA)
• 6) An electrophoresis set-up a machine that
  allows pouring samples in individual wells, and
  exerts electric charge so that materials in the
  samples rise in a manner proportional to their
  molecular weight. Since the terminal base is
  color-marked, it can be easily recognized.
• 7) A computer to read store data (sequences of
  colors each corresponding to a base).

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DNA sequencing(Continued)

• Depending on the purpose, the order (sequence) of
  2 different things need to be determined. Often
  both need to be assessed
• 1- the order of individual bases within short
  fragments of DNA.
• 2- The order of short fragments within a long DNA
  sequence (remenber that genes range in size from
  a few thousand to over 2 million bases)
• By using several restriction enzymes for creating
  fragments of DNA of different lengths, both the
  order of bases within fragments, and the order of
  fragments within larger portions of DNA strands
  can be assessed.
• Computers match specific sequences within
  fragments of different lengths, so that the order
  of fragments is determined.

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Techniques 4) Polymerase Chain Reaction (PCR)

• A method of producing many copies of a small DNA
  sample (so it can be used in experimentation).
• 1) DNA in chromosomes is cut into fragments
  (using restriction enzymes).
• 2) Double strands are separated into single
  strands (using high temperature)
• 3) Primers are introduced
• 4) At lower temperature, DNA polymerase
  synthetizes matching strands to existing single
  strands, by extending primers. The result is new
  double strands.
• 5) The new double strands are separated into
  single strands (high temperature)and the cycle
  continues many times.
• The DNA polymerase of most organisms is very
  similar, but it has small but important
  differences. The DNA polymerase called Taq is
  found in the bacteria Thermus aquaticus, which is
  able to operate in high temperatures. This is the
  commonly used DNA polymerase in PCR.

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Techniques 5) Restriction Fraction Length
Polymorphism (RFLP)

• RFLP is a method used to follow a particular
  sequence of DNA as it is passed on to other cells
  or individuals.
• Based on the fact that within a species, there is
  variation among individuals in the sequence of
  bases at specific sites.
• - This variation is due to differences in the
  genetic makeup of the individuals (the genetic
  information received from each of their patterns)
• - An RFLP is a sequence of DNA that has a
  restriction site on each end with a "target"
  sequence in between. The target is the sequence
  that we are interested in determining whether is
  present or not.
• - The target can be made evident (detected) by
  determining whether a probe will or not attach
  to it. A probe is a single stranded DNA that has
  the complementary bases to the target. If the
  probe attaches, then the individual did have
  the target sequence.
• - Because the probe has been marked with a
  radioactive marker, it can be detected easily on
  a electrophoresis gel.
• RFLP techniques are commonly used in 3 main
  scenarios
• - Medical diagnosis (genetic-based diseases and
  deleterious mutations)
• - paternity, genealogy, identity and criminal
  cases.
• - genetic counseling and family planning.

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RFLP in Medical diagnosis
A)

• RFLP can help to determine if a person carries a
  disease-causing allele.
• 1) Sickle Cell Anemia analysis
• In the diagram, it can be seen that the gene
  responsible for sickle cell anemia has a sequence
  that is longer than normal (B).
• 2) Cystic Fibrosis (hereditary disease caused by
  a recessive allele)

B)

• RFLP analysis would allow seeing
• differences in the bands in samples from
• the individuals involved
• the sickle-cell Gene is longer (molecularly
• heavier) and would show higher bands.
• The samples for cystic fibrosis will show
• either one band (homozygous) or 2 bands
• (heterozygous), which will be located at
• different heights on a gel.

Homozygous Dominant (no disease)
Heterozygous (no disease)
Homozygous Recessive (disease)
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A PCR-RFLP combination assay

• Frequently both techniques are used together, so
  that multiple samples of the same original DNA
  sequence can be assayed in different ways and
  several times (replicates).
• Also, making copies (using PCR), allows saving
  the original sample.
• Generally, the two ends of a gel are not the
  actual samples of interest, but reference bands
  (standards of know molecular weight, or various
  types of control for the techniques used).

Actual samples
Homozygote for allele 2
Homozygote For allele 1
heterozygote
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Techniques RFLP in criminal cases
Blood on suspects clothes
Victims blood
Suspects blood

• A murder trial
• - Is the suspect guilty ?
• - Evidence Blood samples from
• - the victim
• - the suspect
• - blood found in suspects jeans and shirt.
• The suspects blood does not match the blood
  found on the clothes.
• The jeans and shirt have the same type of blood
• The blood on the clothes matches the victims
  blood perfectly.
• The suspect is guilty.

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RFLP in paternity case

• Is Peter the father of Kathy?
• RFLP Analysis 1
• - Because Peter Kathy share a band for this
  particular RFLP, it is possible that Peter is the
  father of Kathy. It becomes necessary to confirm
  it.
• RFLP Analysis 2
• - Since Peter and Kathy do not share one of the
  bands, Peter cannot be the father of Kathy.
• In both cases, the maternity of Mary over Kathy
  has been demonstrated.
• Because RFLP fragments are typically fairly long
  (many bases), the likelihood that 2 individuals
  (that are not identical twins) would share
  several RFLPs is very small.

1)
Peter
Mary
Kathy
2)
Peter
Mary
Kathy
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Techniques 6) Sequencing for determining Single
Nucleotide Polymorphisms (SNPs)

• In SNPs alleles show variations in one or very
  few scattered nucleotides.
• Most SNPs have little or no effect on the
  phenotype, and are therefore harmless. However,
  they are very common in each persons genome.
• Thus, SNPs can become like the individual
  signature of a person.
• SNPs can be used in
• - ascertaining identity
• - studying inheritance patterns
• - studying rates of mutation
• - paternity in certain cases.

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codons ? aminoacids ? proteins

• Each aminoacid is designated by a triplet of
  bases (ATG ? methionine, AAG ? lysine, etc.)
• There are 20 kinds of different aminoacids, but
  there are only 4 bases (A, C, G, T)
• Because the number of triplets that can be made
  from 4 bases (A, C, G, T) is only 64, but there
  are only 20 different aminoacids, there is some
  redundance in the system (each aminoacid is not
  determined by a single unique triplet, but by a
  few triplets). For instance, all of CCA, CCC and
  CCT correspond to the aminoacid proline, and AAG
  or AAA correspond to lysine (in both cases, it
  appears that the third base is meaningless).
• Stop codons Any of the triplets TAA, TGA, or TAG
  serve as stops, which mark the end of a gene, and
  thus the encoding for a protein.

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Mutations Changes in the DNA sequence

• Point mutations
• - One base pair is changed.
• - Most common, often harmless, result in Single
  Nucleotide Polymorphisms (SNPs)
• 1) Missense Results in one aminoacid change. Can
  be serious
• 2) Nonsense Change a codon into a stop codon.
  The resulting protein is shorter, lacking
  aminoacids.
• Frameshift mutations
• - One or two base pairs are inserted or deleted
• - They change every subsequent codon, causing
  large changes. The resulting proteins can be
  shorter, but will definitely have many
  differences, making them non-functional.
• 1) Addition one or more bases are added.
• 2) Deletion One or more bases are deleted.

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Chromosome Rearrangements and Somatic mutations
Segment lost

• Chromosomal mutations (rearrangements) involve
  many bases at a time.
• 1) Deletion A portion of a
• chromosome is lost
• 2) Translocation a portion of chromosome is
  moved to a different chromosome.
• - Some forms of Leukemia (abnormal cancerous
  multiplication of red blood cells) are caused by
  translocation of genes between chromosomes 9 and
  22.
• 3) Inversion A portion of a chromosome changes
  location within the same chromosome.
• Somatic mutations also frequently involve many
  bases at a time. They occur in somatic cells (as
  opposed to reproductive cells).
• - Somatic mutations are not inherited, and may
  or not cause problems. Some can be very serious.
• - Many tumor growths (cancer) are the result of
  somatic mutations.
• - Gene amplification A somatic mutation that is
  part of the normal development in some animals.

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Gene Therapy

• Gene Therapy attempts to treat the genetic
  disorder, not just the symptoms.
• Two main types
• 1) Germ-line Therapy The DNA of the gametes of
  an affected individual can be modified so
  abnormal alleles are not passed on to future
  offspring.
• 2) Somatic cell therapy Corrects the allele
  that causes the genetic disease in the cells that
  express the gene (such as a specific organ of the
  body).
• In both types, genes must be brought into cells,
  and then integrated into the cells DNA.
• Methods of bringing modified genes into cells and
  their DNA
• - Placing the gene in a molecule that can be
  brought in through the cell membrane.
• - Inserting the gene into a virus DNA, and have
  the virus infect the cell, bringing its DNA
  (with the modified gene) into the cells DNA.
  This is what viruses normally do anyway.