‫بسم هللا الرحمن الرحيم‬
‫‪Molecular Biology II‬‬
‫الدكتور ‪ /‬سلمان عبدالعزيز الركيان‬
‫‪Dr. SALMAN ALROKAYAN‬‬
Introduction
‫‪Introduction‬‬
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‫‪DrSALMAN ALROKAYAN‬‬
‫جدول المواضيع لمادة البيولوجيا‬
‫الجزيئية ‪ 446( 2‬كيح)‬
2 ‫جدول المواضيع لمادة البيولوجيا الجزيئية‬
)‫ كيح‬446(
‫عنوان المحاضرات‬
DNA Structure
Genes
Mutations
Mutations
Gene Expression-1
Gene Expression-2
Polymerase Chain
Reaction (PCR)-1
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‫التاريخ‬
‫الرقم‬
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‫عنوان المحاضرات‬
Polymerase Chain
Reaction (PCR)-2
Cloning Vectors -1
Cloning Vectors -2
Exam 1
DNA Cloning-1
DNA Cloning-2
Gel Electrophoresis
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‫التاريخ‬
‫الرقم‬
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‫عنوان المحاضرات‬
Blotting Methods and
Applications-1
Blotting Methods and
Applications-2
DNA Sequencing
Biotechnology
Production of
Recombinant Proteins
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‫التاريخ‬
‫الرقم‬
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‫عنوان المحاضرات‬
Production of
Recombinant Proteins
Final Review
Exam 2
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‫التاريخ‬
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DNA Structure
DNA Structure
A DNA molecule has two strands, held together by the hydrogen bonding
between their bases. As shown in the below figure, adenine can form two
hydrogen bonds with thymine; cytosine can form three 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.
Figure 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.
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The following figure shows an example of base pairing between DNA's two strands.
Due to the specific base pairing, DNA's two strands are complementary to each other.
Figure 2. Schematic drawing of DNA's two strands.
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Genes
Genes
By definition, a gene includes the entire nucleic acid sequence necessary for
the expression of a peptide. Such sequence may be divided into regulatory
region and transcriptional region. The regulatory region could be near or
far from the transcriptional region. The transcriptional region consists of
exons and introns. Exons encode a peptide or functional RNA. Introns will
be removed after transcription
Figure 3. General organization of the DNA sequence. Only the exons
encode a functional peptide or RNA. The coding region accounts for about
3% of the total DNA in a human cell.
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Mutations
Mutations
Mutation refers to the change in a DNA sequence, which may involve only a
few bases or the large-scale chromosome abnormality. This section covers
the small-scale mutations (substitution, deletion, insertion) and the exon
skipping that results from mutation at the splice site.
Substitution
In the substitution mutation, one or more nucleotides are substituted by the
same number of different nucleotides. In most cases, only one nucleotide is
changed. Based on the change in the nucleotide type, the substitution mutation
may be divided into transition and transversion mutations. Based on the
consequence of mutation, the substitution mutation may be grouped into silent,
missense and nonsense mutations.
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Figure 4. The substitution mutation.
(a) Illustration of transition (blue) and transversion (red) mutations. In
the transition mutation, a pyrimidine (C or T) is substituted by another
pyrimidine, or a purine (A or G) is substituted by another purine. The
transversion mutation involves the change from a pyrimidine to a
purine, or vice versa.
(b) Examples of silent, missense and nonsense mutations. The silent
mutation does not produce any change in the amino acid sequence,
the missense mutation results in a different amino acid, and the
nonsense mutation generates a stop signal.
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Deletion
The deletion mutation involves elimination of one or more nucleotides from a DNA
sequence. It may cause frameshift, producing a non-functional protein.
Figure 5. Real examples of deletion mutations which cause diseases.
(a) Deletion of "T" from the sequence "TTTTT" in the CFTR gene.
(b) Deletion of "AT" from the sequence "ATAT" in the CFTR gene.
(c) Deletion of "TTG" from the sequence "TTGTTG" in the FIX gene.
(d) Deletion of "ATAG" from the sequence "ATAGATAG" in the APC gene.
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Insertion
In the insertion mutation, one or more nucleotides are inserted into a sequence. If the
number of inserted bases is not a multiple of 3, it will cause frameshift, resulting in
serious consequences. As shown in the following table, non-frameshift insertions may
also cause diseases.
Table 1. Examples of diseases caused by insertion mutation.
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Exon skipping
Figure 6. Example of exon skipping. Splicing of an intron requires an essential
signal: "GT........AG". If the splice acceptor site AG is mutated (e.g., A to C in this
figure), the splicing machinery will look for the next acceptor site. As a result, the
exon between two introns is also removed.
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Gene Expression
Gene Expression
An organism may contain many types of somatic cells, each with distinct shape and
function. However, they all have the same genome. The genes in a genome do not
have any effect on cellular functions until they are "expressed". Different types of
cells express different sets of genes, thereby exhibiting various shapes and functions.
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"Gene expression" means the production of a protein or a functional RNA from
its gene. Several steps are required:
Transcription: A DNA strand is used as the template to synthesize a RNA
strand, which is called the primary transcript.
RNA processing: This step involves modifications of the primary transcript to
generate a mature mRNA (for protein genes) or a functional tRNA or rRNA.
For RNA genes (tRNA and rRNA), the expression is complete after a
functional tRNA or rRNA is generated. However, protein genes require
additional steps:
Nuclear transport: mRNA has to be transported from the nucleus to the
cytoplasm for protein synthesis.
Protein synthesis: In the cytoplasm, mRNA binds to ribosomes, which
can synthesize a polypeptide based on the sequence of mRNA.
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Polymerase Chain
Reaction (PCR)
Polymerase Chain Reaction
(PCR)
PCR rapidly amplifies a specific fragment of DNA molecule into many billions of
molecules using specific primers. In one application of the technology, small samples of
DNA, such as those found in a strand of hair at a crime scene, can produce sufficient
copies to carry out forensic tests.
Primers are in green color
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Materials required:
1-Two primers, each about 20 bases long with sequence complementary to the sequence
immediately adjacent to the DNA segment of interest.
2-DNA polymerase (e.g., Tag polymerase) which can sustain high temperature (> 60o C)
3-A large number of free deoxynucleotides (dNTPs)
4-The target DNA fragment.
Procedure:
Heat denaturation at about 95oC.
Primers bind to the denatured DNA by base pairing as the temperature is gradually
cooled to about 60o C.
Extend primers with Tag polymerase.
Repeat the above process. The number of copies doubles in each cycle. Typically 20 to
30 cycles are sufficient for effective
DNA amplification.
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Taq polymerase
Originally, DNA polymerase from E. coli was used in the PCR, but this enzyme is heat
sensitive and became denatured when the reaction mixture was heated to separate the
DNA duplexes. This meant that fresh enzyme had to be added to the mixture for each
new cycle. The bacterium Thermus aquaticus however, has a thermostable DNA
polymerase which actually works best at high temperatures, with an optimum functioning
temperature of 72oc, and a reasonable stability at 94oc. Taq polymerase can be added
at the start of one reaction cycle and function through a whole set of amplification cycles.
This has made it possible for the process to become fully automated using thermal
cyclers.
Advantages:
Much faster than using vectors.
Only very small amount of target DNA is needed.
Disadvantages:
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.
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Thermal cyclers
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Gel Electrophoresis
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Cloning Vectors
Cloning Vectors
"Vector" is an agent that can carry a DNA fragment into a host cell. If it is used for
reproducing the DNA fragment, it is called a "cloning vector". If it is used for
expressing certain gene in the DNA fragment, it is called an "expression vector".
Commonly used vectors include plasmid, Lambda phage, cosmid and yeast
artificial chromosome (YAC).
Plasmid
Plasmids are circular, double-stranded DNA molecules that exist in bacteria and in the
nuclei of some eukaryotic cells. They can replicate independently of the host
cell. The size of plasmids ranges from a few kb to near 100 kb
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Figure A typical plasmid vector. It contains a polylinker which can recognize several
different restriction enzymes, an ampicillin-resistance gene (ampr) for selective
amplification, and a replication origin (ORI) for proliferation in the host cell.
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Plasmid vector is made from natural plasmids by removing unnecessary segments and
adding essential sequences. To clone a DNA sample, the same restriction enzyme must
be used to cut both the vector and the DNA sample. Therefore, a vector usually
contains a sequence (polylinker) which can recognize several restriction enzymes so
that the vector can be used for cloning a variety of DNA samples.
A plasmid vector must also contain a drug-resistance gene for selective
amplification. After the vector enters into a host cell, it may proliferate with the host
cell. However, since the transformation efficiency of plasmids in E. coli is very low, most
E. coli cells that proliferate in the medium would not contain the plasmids. Therefore, we
must find a way to allow only the transformed E. coli to proliferate. Typically, antibiotics
are used to kill E. coli cells which do not contain the vectors. The transformed E. coli
cells are protected by the ampicillin-resistance gene (ampr) which can express the
enzyme, b-lactamase, to inactivate the antibiotic ampicillin.
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