WHERE EXACTLY IS THE

CHROMOSOME, CHROMATID,
DNA STRANDS...ETC?
IN THE NUCLEUS OF EACH SOMATIC CELL, THERE IS A "CHROMATIN NETWORK" FORMED OF 46
PACKED CHROMOSOMES PAIRED INTO 23 SETS (22 SOMATIC+1 SEX).EACH CHROMOSOME IS
FORMED OF 2 CHROMATIDS HELD TOGETHER BY A CENTROMERE WHICH MAY BE IN THE MIDDLE
(METACENTRIC) OR SHIFTED FOM THE CENTER (NON-METACENTRIC).
NON-METACENTRIC CHROMOSOMES (SUBMETACENTRIC, ACROCENTRIC AND TELOCENTRIC)HAVE A
SHORT ARM “P” AND LONG ARM “Q”
EACH CHROMATID IS MADE UP OF 2 STRANDS OF DNA (DOUBLE HELIX) TIGHTLY COILED MANY
TIMES AROUND PROTEINS CALLED HISTONES THAT SUPPORT ITS STRUCTURE.              
DNA & GENE STRUCTURE

What is the structure of DNA?

> DNA IN THE CHROMATID  IS FORMED OF 2

STRANDS ( = DOUBLE HELIX POLYNUCLEOTIDE
CHAIN)
> A STRAND IS FORMED OF 5 CARBON ATOM
SUGAR (PENTOSE RING), BASE (ADENINE,
THYMINE, GUANINE, CYTOSINE) AND
PHOSPHATE.
> THE SUGAR AND PHOSPHATE ALTERNATE
TOGETHER TO FORM THE BACKBONE OF THE
DNA MOLECULE IN A WAY THAT THE 5TH
CARBON ATOM OF SUGAR (5’ END) IS
ATTACHED TO THE 3RD CARBON ATOM OF THE
NEXT SUGAR (3’END) BY A PHOSPHATE
GROUP.
REPLICATION OF D.N.A PROCEEDS FROM 5’END TO THE 3’END.
 PURINE BASES (Adenine and guanine) ATTACH TO PYRIMIDINE BASES
(Thymine and cytosine) such that A A=T AND G = C =
 EVERY 3 BASE SEQUENCE FORM A CODON (THEY CODE FOR 1 A.A) –
THE CODE OF SPECIFIC A.A IS UNIVERSAL FOR ALL ORGANISMS
THE 4 DIFFERENT BASES CAN GIVE 64 CODONS,
 SINCE ONLY 20 A.A ARE PRESENT SO MOST A.A HAVE AT LEAST 2 CODONS
EXCEPT TRYPTOPHAN AND METHIONINE – HAVE ONLY ONE CODE-
Locus is the site of a gene on the chromosome.
Alleles are non-identical genes present on the same locus in 2 homologous
chromosomes.
SYNTHESIS OF PROTEINS CODE
AS FOLLOWS:
RNA needs to "see" a signal from the
portion to transcribe, then needs to know
where the protein starts, ends and how
much to make.
 Z-FORM ARE PORTIONS OF D.N.A RICH
IN C-G BONDS THEY MARK AS A SIGNAL
FOR mR.N.A TRANSCRIPTION.
 
 TATA BOXES CODE TO MARK THE
INITIATION SITE FOR TRANSCRIPTION
 
TAG, TGA, TAA CODE TO MARK THE END
OF A TRANSCRIPTION (STOP CODON)
 
CAT BOXES REGULATE THE FREQUENCY
OF TRANSCRIPTION
SOME REGIONS OF THE DNA ARE
FUNCTIONING (EXONS = CODING PORTION,
NEARLY ONLY 2% OF THE TOTAL D.N.A)
OTHERS ARE NON FUNCTIONING (INTRONS
= NON-CODING DNA SEQUENCE)

METHYLATION OF A SEQUENCE =
INACTIVITY

NON-METHYLATION = ACTIVITY

POLYMORPHISM: IT IS THE CHANGE IN

THE DNA NON CODING BASE SEQUENCE
BETWEEN INDIVIDUALS (THUS DO NOT
AFFECT PHENOTYPE).
 
MITOCHONDRIAL CHROMOSOMES:

-         CODE FOR 13 A.A ONLY

-         UNIQUE DNA SEQUENCE – NON

REPETITIVE-.

-         EXCLUSIVELY MATERNAL

-         TGA CODES FOR TRYPTOPHAN

INSTEAD OF BEING A STOP CODON.
HOW ARE GENES IDENTIFIED AND STUDIED?

STUDY AND ANALYSIS OF DNA:

Defintions and terms:

RESTRICTIVE ENDONUCLEASES: Group of enzymes (from bacteria or

fungi) which recognize a short neuceotide sequence in a double stranded
DNA molecule. They cleave (cut) the DNA molecule at each “restriction
site” . About 100 enzymes are identified each specific for a certain
restriction site.

 
 
 DNA LIGASE: enzyme derived from E.Coli which can join DNA
fragments.

VECTORS: e.g Bacteriophage, Viruses and commonly bacteria.

PLASMIDS: Small particles of circular double stranded DNA found in

bacteria. Plasmids are able to replicate in cultures. Foreign DNA can
be inserted into plasmid DNA (Cloned) to replicate.
RECOMBINANT D.N.A: Re-combined Double stranded DNA formed
by cutting and joining of a parent DNA molecule.
REVERSE TRANSCRIPTION: Single stranded DNA formed from mRNA
template, such template is then used to synthesize double stranded
copy DNA (cDNA)
DNA PROBE: Single stranded DNA radiolabelled with Phosphorus
32, it may be:
o  Gene specific probe, obtained from DNA and thus contains both
exons and introns.
o  Complementary DNA probe (cDNA), obtained from mRNA of the
gene under study, thus contain exons (coding sequences) only.
o  Oligonucleotide probe: Synthetic short sequence for a specific
region of a known gene (made of less than 30 base sequences).
FORMATION OF RECOMBINANT
D.N.A:
The circular DNA of plasmid is opened by restrictive endonuclease
which is the same used to fragment the donor DNA, then the donor
DNA fragment –To be studied- is inserted into the circle where it will
attach to the properly cut base sequence –Recombined- (since same
enzyme must be used), then the circle of plasmid is reformed by ligase
enzyme, now it is called Hybrid plasmid or Hybrid DNA.
The Hybrid plasmid (containing the plasmid and foreign DNA) is
introduced into a bacteria that will multiply thus replicating the hybrid
plasmid.
Replication of hybrid plasmid allows the isolation of large quantities of
cloned DNA –reobtained after replication by the same restrictive
endonuclease- and now the base sequence of the DNA desired
fragment identified by southern blotting, the gene can be studied or
used to produce the gene product.  
 
MAKING RECOMBINANT DNA (RDNA): AN
OVERVIEW
• Treat DNA from both sources with the same restriction endonuclease
(BamHI in this case).

• BamHI cuts the same site on both molecules

5' GGATCC 3'
3' CCTAGG 5'

• The ends of the cut have an overhanging piece of single-stranded DNA.

• These are called "sticky ends" because they are able to base pair with
any DNA molecule containing the complementary sticky end.

• In this case, both DNA preparations have complementary sticky ends

and thus can pair with each other when mixed.

• DNA ligase covalently links the two into a molecule of recombinant

DNA.
 To be useful, the recombinant molecule must be replicated many times to provide material
for analysis, sequencing, etc. Producing many identical copies of the same recombinant
molecule is called cloning. Cloning can be done in vitro, by a process called the
polymerase chain reaction (PCR). Here, however, we shall examine how cloning is done
in vivo.
Cloning in vivo can be done
 in unicellular microbes like E. coli
 unicellular eukaryotes like yeast and
 in mammalian cells grown in tissue culture.

In every case, the recombinant DNA must be taken up by the cell in a

form in which it can be replicated and expressed. This is achieved by
incorporating the DNA in a vector. A number of viruses (both bacterial
and of mammalian cells) can serve as vectors. But here let us examine
an example of cloning using E. coli as the host and a plasmid as the
vector.
PLASMIDS
Plasmids are molecules of DNA that are found in bacteria separate
from the bacterial chromosome.

They:

 are small (a few thousand base pairs)

 usually carry only one or a few genes
 are circular
 have a single origin of replication

Plasmids are replicated by the same machinery that replicates the

bacterial chromosome. Some plasmids are copied at about the same rate
as the chromosome, so a single cell is apt to have only a single copy of
the plasmid. Other plasmids are copied at a high rate and a single cell
may have 50 or more of them.
Genes on plasmids with high numbers of copies are usually expressed at
high levels. In nature, these genes often encode proteins (e.g.,
enzymes) that protect the bacterium from one or more antibiotics.
SOME RECOMBINANT DNA PRODUCTS BEING USED
IN HUMAN THERAPY

 insulin for diabetics

 factor VIII for males suffering from hemophilia A
 factor IX for hemophilia B human growth hormone (HGH)
 erythropoietin (EPO) for treating anemia
 several types of interferon
 several interleukins
 granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating
the bone marrow after a bone marrow transplant
tissue plasminogen activator (TPA) for dissolving blood clots
 adenosine deaminase (ADA) for treating some forms of severe combined
immunodeficiency (SCID)
 parathyroid hormone
 several monoclonal antibodies
 hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus
FORMATION OF A PROBE:
 Same as above till the recombinant DNA is
obtained, then it is labeled with a
radioactive isotope. Since it is double
stranded then separation of strands is done
by heat to obtain a single stranded
radiolabelled DNA probe.
IDENTIFICATION OF DNA
FRAGMENTS:

 When restrictive endonuclease is used the DNA

split to multiple mixed fragments, they are then
separated by gel electrophoresis. The gel is then
blotted on nitrocellulose paper.

 Now we have separate solid DNA fragments. A

probe is then added to bind to its matching fragment
which can then be visualized by autoradiography.
SEPARATION OF A SUBSTANCE BY
ELECTROPHORETIC GEL THEN
BLOTTING IT ON NITROCELLULOSE
PAPER TO TURN IT TO A SOLID
FORM IS TERMED “BLOTTING”.
 If the substance is DNA it is termed Southern
Blotting.

 If the substance is RNA it is termed Northern

blotting.

 If the substance is protein it is termed Western

blotting.
POLYMERASE CHAIN REACTION
(PCR):
  In presence of polymerase enzyme (heat resistant) separation
of a double stranded DNA (parentral strands) by heat at 95
degrees.

o       Annealing: Synthetic pair of oligonucleotide probes are

allowed to attach to their matcing base sequences on the
separated DNA helicals.

o       Since DNA polymerase is not denaturated by heat, its

presence will allow the small synthesized oligonucleotide probe
to extend along the specific DNA fragment – sort of replication-.

o       This 3 step cycle is repeated 25 to 35 times.

 
PROCEDURE
 Typically, PCR consists of a series of 20-40 repeated
temperature changes, called cycles, with each cycle
commonly consisting of 2-3 discrete temperature steps,
usually three .

The cycling is often preceded by a single temperature

step (called hold) at a high temperature (>90°C), and
followed by one hold at the end for final product
extension or brief storage.

The temperatures used and the length of time they

are applied in each cycle depend on a variety of
parameters.

These include the enzyme used for DNA synthesis, the

concentration of divalent ions and dNTPs in the
reaction, and the melting temperature (Tm) of the
primers.
INITIALIZATION STEP: This step consists of heating the reaction
to a temperature of 94–96 °C (or 98 °C if extremely thermostable
polymerases are used), which is held for 1–9 minutes. It is only
required for DNA polymerases that require heat activation by
hot-start PCR.[9]

DENATURATION STEP: This step is the first regular cycling event

and consists of heating the reaction to 94–98 °C for 20–30 seconds. It
causes DNA melting of the DNA template by disrupting the hydrogen
bonds between complementary bases, yielding single-stranded DNA
molecules.
ANNEALING STEP: The reaction temperature is lowered to 50–
65 °C for 20–40 seconds allowing annealing of the primers to the
single-stranded DNA template. Typically the annealing temperature
is about 3-5 degrees Celsius below the Tm of the primers used.
Stable DNA-DNA hydrogen bonds are only formed when the primer
sequence very closely matches the template sequence. The
polymerase binds to the primer-template hybrid and begins DNA
synthesis.
EXTENSION/ELONGATION STEP: The temperature at this step
depends on the DNA polymerase used; Taq polymerase has its
optimum activity temperature at 75–80 °C,and commonly a
temperature of 72 °C is used with this enzyme. At this step the DNA
polymerase synthesizes a new DNA strand complementary to the
DNA template strand by adding dNTPs that are complementary to
the template in 5' to 3' direction, condensing the 5'-phosphate group
of the dNTPs with the 3'-hydroxyl group at the end of the nascent
(extending) DNA strand.

FINAL ELONGATION: This single step is occasionally performed at a

temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to
ensure that any remaining single-stranded DNA is fully extended.

FINAL HOLD: This step at 4–15 °C for an indefinite time may be

employed for short-term storage of the reaction.