Molecular Biology II Dr. SALMAN ALROKAYAN بسم الله الرحمن الرحيم الدكتور /...

Molecular Biology II

Dr. SALMAN ALROKAYAN

الرحيم الرحمن الله الرحيم بسم الرحمن الله بسم

الركيان / عبدالعزيز سلمان الركيان / الدكتور عبدالعزيز سلمان الدكتور

Introduction

الركيان عبدالعزيز بن سلمان الدكتور 3DrSALMAN ALROKAYAN

Introduction

لمادة المواضيع جدولالجزيئية 2البيولوجيا

(446 ) كيح


البيولوجيا لمادة المواضيع جدول( 446 )2الجزيئية كيح

المحاضرات الرقم التاريخ عنوان

DNA Structure 1

Genes 1

Mutations 2

Mutations2

Gene Expression-1 3

Gene Expression-23

Polymerase Chain Reaction )PCR(-1

4



Polymerase Chain Reaction )PCR(-2

4

Cloning Vectors -1 \5

Cloning Vectors -2 5

Exam 1 6

DNA Cloning-1 7

DNA Cloning-2 7

Gel Electrophoresis 8



Blotting Methods and Applications-1

9

Blotting Methods and Applications-2

9

DNA Sequencing 10

Biotechnology 11

Production of Recombinant Proteins

11



Production of Recombinant Proteins

11

Final Review 12

Exam 2 13

DNA Structure


DNA Structure

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.

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.


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

Figure 2. Schematic drawing of DNA's two strands.

Due to the specific base pairing, DNA's two strands are complementary to each other.


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.

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.

Mutations

SubstitutionIn 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.


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.


DeletionThe 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.


InsertionIn 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.


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.

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.


"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.

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


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 .


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 .

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.

Taq polymerase


Thermal cyclers


Gel Electrophoresis

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( .

PlasmidPlasmids 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


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.


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.

Molecular Biology II Dr. SALMAN ALROKAYAN بسم الله الرحمن الرحيم الدكتور /...

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Transcript of Molecular Biology II Dr. SALMAN ALROKAYAN بسم الله الرحمن الرحيم الدكتور /...