Archive for February 9th, 2013

Gene Therapy

Gene Therapy.


Gene Therapy

What is gene therapy?

Genes, which are carried on chromosomes, are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. Although genes get a lot of attention, it’s the proteins that perform most life functions and even make up the majority of cellular structures. When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result.

Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:

  • A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.
  • An abnormal gene could be swapped for a normal gene through homologous recombination.
  • The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.
  • The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

How does gene therapy work?

In most gene therapy studies, a “normal” gene is inserted into the genome to replace an “abnormal,” disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient’s target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes.

Target cells such as the patient’s liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state. See a diagram depicting this process.

Some of the different types of viruses used as gene therapy vectors:

  • Retroviruses – A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus.
  • Adenoviruses – A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus.
  • Adeno-associated viruses – A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19.
  • Herpes simplex viruses – A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.

Besides virus-mediated gene-delivery systems, there are several nonviral options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA.

Another nonviral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell’s membrane.

Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell. This delivery system tends to be less effective than other options.

Researchers also are experimenting with introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 –not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the body’s immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell.


PCR (Polymerase Chain Reaction)

PCR is a cell-free method of DNA cloning.  It is much faster and more sensitive than cell-based cloning.

Figure 9-E-1.  Polymerase Chain Reaction (PCR).  Primers are in green color.

Materials required:

  • Two primers, each about 20 bases long with sequence complementary to the sequence immediately adjacent to the DNA segment of interest.
  • DNA polymerase (e.g., Tag polymerase) which can sustain high temperature (> 60o C)
  • A large number of free deoxynucleotides (dNTPs)
  • The target DNA fragment.


  1. Heat denaturation at about 95oC.
  2. Primers bind to the denatured DNA by base pairing as the temperature is gradually cooled to about 60o C.
  3. Extend primers with Tag polymerase.
  4. Repeat the above process.  The number of copies doubles in each cycle.  Typically 20 to 30 cycles are sufficient for effective DNA amplification.


  • Much faster than using vectors.
  • Only very small amount of target DNA is needed.


  • To synthesize primers, we need to know the sequence flanking the DNA segment of interest.
  • Applies only to short DNA fragments, typically less than 5 kb.


D.N.A. Structure



Figure 3-B-1.  Computer model of base pairing in DNA.  In a normal DNA molecule, adenine (A) is paired with thymine (T), guanine (G) is paired with cytosine (C).  The uracil (U) of RNA can also pair with adenine (A), since U differs from T by only a methyl group located on the other side of hydrogen bonding.


A DNA molecule has two strands, held together by the hydrogen bonding between their bases.  As shown in the above figure, adenine can form two hydrogen bonds with thymine; cytosine can formthree hydrogen bonds with guanine.  Although other base pairs [e.g., (G:T) and (C:T) ] may also form hydrogen bonds, their strengths are not as strong as (C:G) and (A:T) found in natural DNA molecules.

The following figure shows an example of base pairing between DNA’s two strands.

Figure 3-B-2.  Schematic drawing of DNA’s two strands.


Due to the specific base pairing, DNA’s two strands are complementary to each other.  Hence, the nucleotide sequence of one strand determines the sequence of another strand.  For example, in Figure 3-B-2, the sequence of the two strands can be written as

5′ -ACT- 3′

3′ -TGA- 5′

Note that they obey the (A:T) and (C:G) pairing rule.  If we know the sequence of one strand, we can deduce the sequence of another strand.  For this reason, a DNA database needs to store only the sequence of one strand.  By convention, the sequence in a DNA database refers to the sequence of the 5′ to 3′ strand (left to right).


D.N.A. Replication:

There is a major difference between DNA polymerase and RNA polymerasethe RNA polymerase can synthesize a new strand whereas the DNA polymerase can only extend an existing strand.  Therefore, to synthesize a DNA molecule, a short RNA molecule (~ 5 – 12 nucleotides) must be synthesize first by a special enzyme.  The initiating RNA molecule is known as a primer, and the enzyme is called primase.

In addition to DNA polymerase and primase, DNA replication requires helicase and single strand binding protein (SSB protein).  The role of helicase is to unwind the duplex DNA.  SSB proteins can bind to both separated strands, preventing them from annealing (reconstitution of double-stranded DNA from single strands).

The replication mechanisms in both bacteria and eukaryotes are similar.  However, eukaryotic DNA polymerases do not contain a subunit similar to the E. coli b subunit.  They use a separate protein called proliferating cell nuclear antigen (PCNA) to clamp the DNA.


b7-b-2.GIF (29118 bytes)

Figure 7-B-2.  Structure of PCNA which is formed by three identical subunits.  PDB ID = 1AXC.


DNA polymerases can extend nucleic acid strands only in the 5′ to 3′ direction.  However, in the direction of a growing fork, only one strand is from 5′ to 3′.  This strand (the leading strand) can be synthesized continuously.  The other strand (the lagging strand), whose 5′ to 3′ direction is opposite to the movement of a growing fork, should be synthesized discontinuously.


Figure 7-B-3.  Steps in the synthesis of the lagging strand.

(a) Comparison between the leading strand and the lagging strand.

(b) The primase first synthesizes a new primer which is about 10 nucleotides in length.  The distance between two primers is about 1000-2000 nucleotides in bacteria, and about 100-200 nucleotides in eukaryotic cells.

(c)  DNA polymerase elongates the new primer in the 5′ to 3′ direction until it reaches the 5′ end of a neighboring primer.  The newly synthesized DNA is called an Okazaki fragment.

(d) In E. coli, DNA polymerase I has the 5′ to 3′ exonuclease activity, which is used to remove a primer.

(e) DNA ligase joins adjacent Okazaki fragments.

The whole lagging strand is synthesized by repeating steps (b) to (e).


Mendelian Genetics

Mendelian Genetics.

Mendelian Genetics

Mendelian Genetics

Mendel 1862

Mendel 1868 Mendel 1880
1862 1868 1880

Genetic Terminology:

  • Trait – any characteristic that can be passed from parent to offspring
  • Heredity – passing of traits from parent to offspring
  • Genetics – study of heredity
  • Alleles – two forms of a gene (dominant & recessive)
  • Dominant – stronger of two genes expressed in the hybrid; represented by a capital letter (R)
  • Recessive – gene that shows up less often in a cross; represented by a lowercase letter (r)
  • Genotype – gene combination for a trait (e.g. RR, Rr, rr)
  • Phenotype – the physical feature resulting from a genotype (e.g. tall, short)
  • Homozygous genotype – gene combination involving 2 dominant or 2 recessive genes (e.g. RR or rr); also called pure 
  • Heterozygous genotype – gene combination of one dominant & one recessive allele    (e.g. Rr); also called hybrid
  • Monohybrid cross – cross involving a single trait
  • Dihybrid cross – cross involving two traits
  • Punnett Square – used to solve genetics problems

Blending Concept of Inheritance:

  • Accepted before Mendel’s experiments
  • Theory stated that offspring would have traits intermediate between those of its parents such as red & white flowers producing pink
  • The appearance of red or white flowers again was consider instability in genetic material
  • Blending theory was of no help to Charles Darwin’s theory of evolution 
  • Blending theory did not account for variation and could not explain species diversity
  • Particulate theory of Inheritance, proposed by Mendel, accounted for variation in a population generation after generation
  • Mendel’s work was unrecognized until 1900

Gregor Mendel:

  • Austrian monk

  • Studied science & math at the University of Vienna

  • Formulated the laws of heredity in the early 1860’s

  • Did a statistical study of  traits in garden peas over an eight year period.

Why peas, Pisum sativum?

  • Can be grown in a small area

  • Produce lots of offspring

  • Produce pure plants when allowed to self-pollinate several generations

  • Can be artificially cross-pollinate

Picture of Pisum sativum

Mendel’s Experiments:

  • Mendel studied simple traits from 22 varieties of  pea plants (seed color & shape, pod color & shape, etc.)

  • Mendel traced the inheritance of individual traits & kept careful records of numbers of offspring

  • He used his math principles of probability to interpret results

  • Mendel studied pea traits, each of which had a dominant & a recessive form (alleles)

  • The dominant (shows up most often) gene or allele is represented with a capital letter, & the recessive gene with alower case of that same letter (e.g. B, b)

  • Mendel’s traits included:

            a. Seed shape —  Round (R) or Wrinkled (r)
            b. Seed Color —- Yellow (Y) or  Green (y)
            c. Pod Shape — Smooth (S) or wrinkled (s)
            d. Pod Color —  Green (G) or Yellow (g)
            e. Seed Coat Color —  Gray (G) or White (g)
            f. Flower position — Axial (A) or Terminal (a)
            g. Plant Height — Tall (T) or Short (t)
            h. Flower color — Purple (P) or white (p)

  •  Mendel produced pure strains by allowing the plants to self-pollinate for several generations
  • These strains were called the Parental generation or P1 strain
  • Mendel cross-pollinated two strains and tracked each trait through two
    generations (e.g. TT  x  tt )

                  Trait – plant height

                  Alleles – T tall, t short

    P1 cross    TT  x  tt

    genotype      —    Tt
    t t phenotype    —    Tall
    T Tt Tt genotypic ratio –all alike
    T Tt Tt phenotypic ratio- all alike

  • The offspring of this cross were all hybrids showing only the dominant trait & were called the First Filial or F1generation

  • Mendel then crossed two of his F1 plants and tracked their traits; known as an F1 cross

              Trait – plant height

              Alleles – T tall, t short

F1 cross    Tt  x  Tt

genotype      —    TT, Tt, tt
T t phenotype    —    Tall & short
T TT Tt genotypic ratio —1:2:1
t Tt tt phenotypic ratio- 3:1

  • When 2 hybrids were crossed, 75% (3/4) of the offspring showed the dominant trait & 25% (1/4) showed the recessive trait; always a 3:1 ratio

  • The offspring of this cross were called the F2 generation

  • Mendel then crossed a pure & a hybrid from his Fgeneration; known as an F2 or test cross

Trait   –  Plant Height

Alleles – T  tall, t  short

F2 cross       TT  x Tt

F2 cross       tt  x Tt

T t T t
T TT Tt t Tt tt
T TT Tt t Tt tt

          genotype – TT, Tt

          genotype – tt, Tt

          phenotype  –  Tall

          phenotype  –  Tall & short

          genotypic ratio  – 1:1

          genotypic ratio  – 1:1

          phenotypic ratio – all alike

          phenotypic ratio – 1:1
  • 50% (1/2) of the offspring in a test cross showed the same genotype of one parent & the other 50% showed the genotype of the other parent; always a 1:1 ratio

Problems: Work the P1, F1, and both F2 crosses for all of the other pea plant traits & be sure to include genotypes, phenotypes, genotypic & phenotypic ratios.

  • Mendel also crossed plants that differed in two characteristics (Dihybrid Crosses)
    such as 
    seed shape & seed color

  • In the P1 cross, RRYY  x  rryy, all of the F1 offspring showed only the dominant form for both traits; all hybrids, RrYy

Traits:      Seed Shape & Seed Color

Alleles:     R round                Y yellow
r wrinkled             y green

 P1 Cross:     RRYY          x     r r yy  








     Round yellow seed

Genotypic ratio:

     All alike

Phenotypic ratio:

     All Alike
  • When Mendel crossed 2 hybrid plants (F1 cross), he got the following results

Traits:       Seed Shape & Seed Color

Alleles:     R round                Y yellow
r wrinkled             y green

     F1 Cross:     RrYy           x     RrYy                    

RY Ry rY ry








r rYY

r rYy


r rYy

r ryy

Genotypes Genotypic Ratios


Phenotypic Ratios


Round yellow seed


RRYy 2
RrYY 2
RrYy 4
RRyy 1

Round green seed

Rryy 2
r rYY 1

Wrinkled yellow seed

r rYy 2
r ryy 1

Wrinkled green seed


Problems: Choose two other pea plant traits and work the P1 and F1 dihybrid crosses. Be sure to show the trait, alleles, genotypes, phenotypes, and all ratios. 

Results of Mendel’s Experiments:

  • Inheritable factors or genes are responsible for all heritable characteristics

  • Phenotype is based on Genotype

  • Each trait is based on two genes, one from the mother and the other from the father

  • True-breeding individuals are homozygous ( both alleles) are the same

  • Law of Dominance states that when different alleles for a characteristic are inherited (heterozygous), the trait of only one (the dominant one) will be expressed. The recessive trait’s phenotype only appears in true-breeding (homozygous) individuals

Trait: Pod Color
Genotypes: Phenotype:
GG Green Pod
Gg Green Pod
gg Yellow Pod
  • Law of Segregation states that each genetic trait is produced by a pair of alleles which separate (segregate) during reproduction


R r
  • Law of Independent Assortment states that each factor (gene) is distributed (assorted) randomly and independently ofone another in the formation of gametes


RY Ry rY ry

Other Patterns of Inheritance:

  • Incomplete dominance occurs in the heterozygous or hybrid genotype where the 2 alleles blend to give a different phenotype

  • Flower color in snapdragons shows incomplete dominance whenever a red flower is crossed with a white flower to produce pink flowers

  • In some populations, multiple alleles (3 or more) may determine a trait such as in ABO Blood type

  • Alleles A & B are dominant, while O is recessive

Genotype Phenotype
  • Polygenic inheritance occurs whenever many variations in the resulting phenotypes such as in hair, skin, & eye color

  • The expression of a gene is also influenced by environmental factors (example: seasonal change in fur color)