1. Molecular Biology (MLMB-201) Lecturer: Dr. Mohamed Salah El-Din Department of Medical Laboratory Technology Faculty of Allied Medical Science

2. Intended Learning Outcomes (ILO’s): Molecular biology course provides an overview of the molecular basis to cell structure and function. This course focuses on the structure, biosynthesis and function of DNA and RNA on the molecular level and how these interact among themselves and with proteins. Molecular biology techniques are essential for modern biological and medical research. This course will give you an introduction to DNA and RNA standard techniques. Student will have basic knowledge of: Cell organization. DNA structure and function. DNA Extraction. RNA structure and function. RNA Extraction. Gene expression and protein biosynthesis. Agarose gel electrophoresis for DNA/RNA; and SDS-PAGE for protein. Polymerase Chain Reaction (PCR) – Theory, Types, Application. Gene library and screening DNA sequencing

3. Gene library and screening

4. I 1 Genomic libraries I 2 cDNA libraries I 3 Screening procedures Gene libraries and screening

5. I1 Genomic libraries  Representative gene libraries Gene libraries made from genomic DNA are called genomic libraries and those made from complementary DNA are known as cDNA libraries. The latter lack nontranscribed genomic sequences(repetitive sequences, etc.). Good gene libraries are representative of the starting material and have not lost certain sequences due to cloning artifacts.  Size of library A gene library must contain a certain number of recombinant for there to be a high probability of it containing any particular sequence. This value can be calculated if the genome size and the average size of the insert in the vector are known.  Genomic DNA For making libraries, genomic DNA, usually prepared by protease digestion and phase extraction, is fragmented randomly by physical shearing or restriction enzyme digestion to give a size range appropriate for the chosen vector. Often combination of restriction enzymes are used to partially digest the DNA.  Vectors Plasmids, λ pahge, cosmid, BAC or yeast artificial chromosome vectors can be used to construct genomic libraries, the choice depending on the genome size. The upper size limit of these vectors is about 10,23,45,350 and 1000 kb respectively. The genomic DNA fragments are ligated to the prepared vector molecule using T4 DNA ligase.

6. I 2 cDNA libraries  mRNA isolation, purification and fractionation mRNA can be readily isolated from lysed eukaryotic cells by adding magnetic beads which have oligo(dT) covalently attached. The mRNA binds to the oligo(dT) via its poly(A) tail and thus be isolated from the solution. The integrity of an mRNA preparation can be checked by translation in a wheat germ extract or reticulocyte lysate and then visualizing the translation products by polyacrylaminde gel electrophoresis. Integrity can also be studied using gel electrophoresis, which allows the mRNA to be size fractionated by recovering chosen regions of the gel lane. Specific sequences can be removed from the mRNA by hybridization. mRNA can be readily isolated from lysed eukaryotic cells by adding magnetic beads which have oligo(dT) covalently attached. The mRNA binds to the oligo(dT) via its poly(A) tail and thus be isolated from the solution. The integrity of an mRNA preparation can be checked by translation in a wheat germ extract or reticulocyte lysate and then visualizing the translation products by polyacrylaminde gel electrophoresis. Integrity can also be studied using gel electrophoresis, which allows the mRNA to be size fractionated by recovering chosen regions of the gel lane. Specific sequences can be removed from the mRNA by hybridization.

7.  Synthesis of cDNA In the first synthesis, reverse transcriptase is used to make a cDNA copy of the mRNA by exetending a primer, usually oligo(dT), by the addition of deoxyribounucletides to 3’-end. Synthesis can be detected by trace labeling. 3’-Tailing of the first strand cDNA using terminal transferase makes full-length second strand synthesis easier. Reverse transcriptase or Klenow enzyme can extend a primer [e.g.oligo(dG)] annealed to a homopolymeric tail [e.g.oligo(dC)] to synthesize second strand cDNA.  Treatment of cDNA ends To avoid blunt end ligation of cDNA to vector, linkers are usually added to the cDNA after the ends have been repaired(blunted) using a single strand-specific nuclease followed by Klenow enzyme. The cDNA may also be methylated to keep it from being digested when the added linkers are cleaved by a restriction enzyme. Adaper molecules can be used as an alternative to linkers.  Ligation to vector The vector is usually dephosphorylated using alkaline phosphatase to prevent self-ligation, and so promote the formation of recombinant molecules. Plasmid or phage vectors can be used to make cDNA libraries, but the phage λgt11 is preferred for the cnstruction of expression libraries.

8. cDNA cloning. (a) First and second strand synthesis; (b) end preparation and linker addition to duplex cDNA.

9. I3 Screening procedures  Screening Screening to isolate one particular clone from a gene library routinely involves using a nucleic acid probe for hybridization. The probe will bind to its complementary sequence allowing the required clone to be identified.  Colony and plaque hybridization A copy of the position of colonies or plaques on a petri is made on the surface of a membrane, which is then incubated in a solution of labeled probe. After hybridization and washing, the location of the bound label is determined. The group of colonies/plaques to which the label has bound is diluted and re-plated in subsequent rounds of screening until an individual clone is obtained.

10.  Expression screening One-sixth of the clones in a cDNA expression libraries will produce their encoded polypeptide as a fusion protein with the vector-encodeed β-galactosidase. Antibidies to the desired protein can be used to screen the library in a process similar to plaque hybridization to obtain particular clone.  Hybrid arrest and release cDNA clones can be hybridized to mRNA preparations to prevent, or arrest, the subsequent translation of some mRNA species. Alternatively, those mRNA hybridized to the cDNA clones can be purified, released from the cDNA and translated to identify the polypeptides encoded.  Chromosome walking This is the repeated screening of genomic libraries to obtain overlapping clones and hence build up a collection of clones covering part of a chromosome. It involves using the ends of the inserts as probes for subquent rounds of screening. Chromosome jumping is a similar process.

11. Screening by plaque hybridization

12. Chromosome walking

13. DNA Sequencing  Describe the rationale behind the Sanger dideoxy sequencing technique.  What are the necessary components of sequencing reactions?  How is the radiogram interpreted in sequencing reactions?  In what ways are the sequencing reactions different in an automated DNA procedure?  What types of information does DNA sequencing reveal?

14. Dideoxy DNA sequencing of a theoretical DNA fragment. (The “number of nucleotides” in the gel analysis refers to nucleotides added to the primer during new DNA synthesis.)

16. Can you read this sequence? TRY IT A 5’ Read from the bottom up Autoradiogram of a dideoxy sequencing gel. The letters over the lanes (A, C, G, and T) correspond to the particular dideoxy nucleotide used in the sequencing reaction analyzed in a given lane.

17. Results of automated DNA sequence analysis using fluorescent dyes. The procedure is described in the text. The automated sequencer generates the curves shown in the figure from the fluorescing bands on the gel. The colors are generated by the machine and indicate the four bases: A is green, G is black, C is blue, and T is red. Where bands cannot be distinguished clearly, an N is listed.

18. PCR  Describe the rationale behind the polymerase chain reaction (PCR) technique.  What are the necessary components of PCR reactions?  Why must the DNA polymerase be highly thermally stable?  What are the temperatures of the thermal cycle, and what occurs at each of these temperature?  What are some advantages and limitations of PCR?  What are some applications of PCR?

19.  What is RT-PCR, and in what ways is it different from PCR reactions?  Why is PCR poorly quantitative?  How has RT-PCR been made to be quantitative?

20. The polymerase chain reaction (PCR) for selective amplification of DNA sequences.

21. Practice 11. The diagram above (Figure 1) shows the products from a Sanger method of enzymatic extension sequencing of DNA. What is the sequence of the TEMPLATE strand? a.5’-GACCGCT-3’b. 5’-TCGCCAG-3’ c. 5’-AGCGGTC-3’ d. 5’-CTGGCGA-3’

22. Assignment: As a part of the semester activity, a group of students is selected every week to prepare a short seminar about his/her point of interest in one of the lecture topics. That to be discussed and evaluated during the next lecture.