Molecular Techniques By Dr. Reem Sallam

Objectives To Identify molecular techniques that are currently in clinical practice To understand the principle, types and medical applications of PCR To understand the use of restriction endonucleases for the production of recombinant DNA and recombinant proteins To understand the principle of blotting techniques and their medical applications Reem Sallam, MD, PhD

Lecture Outlines Molecular techniques: an overview PCR Restriction endonucleases, recombinant DNA & recombinant proteins Blotting techniques: Southern, northern, western techniques The use of Southern blotting for the detection of mutations Reem Sallam, MD, PhD

Molecular techniques: Overview Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

This knowledge has led to the development of methods for In the past, efforts to understand genes and their expression have been confounded by the immense size and complexity of human deoxyribonucleic acid (DNA). The human genome contains DNA with approximately three billion (109) base pairs that encode 20,000 to 30,000 genes located on 23 pairs of chromosomes. It is now possible to determine the nucleotide sequence of long stretches of DNA, and the entire sequence of the human genome has been determined. This effort (called the Human Genome Project) was made possible by several techniques that have already contributed to our understanding of many genetic diseases. These include: The discovery of restriction endonucleases that permit the dissection of huge DNA molecules into defined fragments. The development of cloning techniques, providing a mechanism for amplification of specific nucleotide sequences. The ability to synthesize specific probes, which has allowed the identification and manipulation of nucleotide sequences of interest. These and other experimental approaches have permitted the identification of both normal and mutant nucleotide sequences in DNA. This knowledge has led to the development of methods for the prenatal diagnosis of genetic diseases, and initial successes in the treatment of patients by gene therapy. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Restriction endonucleases One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded (ds) DNA into smaller, more manageable fragments, has opened the way for DNA analysis. Because each enzyme cleaves DNA at a specific nucleotide sequence, restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

A. Specificity of restriction endonucleases Restriction endonucleases recognize short stretches of DNA (generally four or six base pairs) that contain specific nucleotide sequences. These sequences, which differ for each restriction endonuclease, are palindromes, that is, they exhibit twofold rotational symmetry. This means that, within a short region of the double helix, the nucleotide sequence on the “top” strand, read 5′→3′, is identical to that of the “bottom” strand, also read in the 5′→3′ direction. Therefore, if you turn the page upside down—that is, rotate it 180 degrees around its axis of symmetry—the structure remains the same. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

C. “Sticky” and “blunt” ends Restriction enzymes cleave DNA so as to produce a 3′-hydroxyl group on one end and a 5′-phosphate group on the other. Some restriction endonucleases, such as TaqI, form staggered cuts that produce “sticky” or cohesive ends—that is, the resulting DNA fragments have single-stranded (ss) sequences that are complementary to each other. Other restriction endonucleases, such as HaeIII, produce fragments that have “blunt” ends that are double-stranded and therefore do not form hydrogen bonds with each other. Using the enzyme DNA ligase, sticky ends of a DNA fragment of interest can be covalently joined with other DNA fragments that have sticky ends produced by cleavage with the same restriction endonuclease. Another ligase, encoded by bacteriophage T4, can covalently join blunt-ended fragments. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

B. Nomenclature A restriction enzyme is named according to the organism from which it was isolated. The first letter of the name is from the genus of the bacterium. The next two letters are from the name of the species. An additional subscript letter indicates the type or strain, and a final number is appended to indicate the order in which the enzyme was discovered in that particular organism. For example, HaeIII is the third restriction endonuclease isolated from the bacterium Haemophilus aegyptius Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

D. Restriction sites A DNA sequence that is recognized by a restriction enzyme is called a restriction site. These sites are recognized by restriction endonucleases that cleave DNA into fragments of different sizes. For example, an enzyme that recognizes a specific four-base-pair sequence produces many cuts in the DNA molecule. In contrast, an enzyme requiring a unique sequence of six base pairs produces fewer cuts and, hence, longer pieces. Hundreds of these enzymes, having different cleavage specificities (varying in both nucleotide sequences and length of recognition sites), are commercially available as analytic reagents. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Specificity of RE Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Specificity of RE; Sticky & Blunt ends Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

The use of DNA ligase to form a recombinant DNA from restriction fragments If restriction fragments are with sticky ends If restriction fragments are with blunt ends: use bacteriophage T4 ligase Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Polymerase Chain Reaction PCR

PCR, Overview The polymerase chain reaction (PCR) is a test tube method for amplifying a selected DNA sequence that does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source—bacterial, viral, plant, or animal Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

A. Steps of a PCR PCR uses DNA polymerase to repetitively amplify targeted portions of DNA. Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase in DNA with repeated cycles of amplification. The amplified DNA sequence can then be analyzed by gel electrophoresis, Southern hybridization, or direct sequence determination. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

A.1 Primer construction: It is not necessary to know the nucleotide sequence of the target DNA in the PCR method. However, it is necessary to know the nucleotide sequence of short segments on each side of the target DNA. These stretches, called flanking sequences, bracket the DNA sequence of interest. The nucleotide sequences of the flanking regions are used to construct two, single-stranded oligonucleotides, usually 20–35 nucleotides long, which are complementary to the respective flanking sequences. The 3′-hydroxyl end of each primer points toward the target sequence. These synthetic oligonucleotides function as primers in PCR reactions. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Steps in one cycle of PCR The 3′-OH end of each primer points toward the target sequence. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Multiple cycles of PCR Lippincott’s Illustrated Review of Biochemistry , 4th Edition, Chapter 33

A-2 Denature the DNA A-3 Annealing of primers to ssDNA A-2 Denature the DNA: The DNA to be amplified is heated to separate the ds target DNA into ss. A-3 Annealing of primers to ssDNA: The separated strands are cooled and allowed to anneal to the two primers (one for each strand). Lippincott’s Illustrated Review of Biochemistry 4th Edition, Chapter 33

A-4 Chain extension DNA polymerase and dNTPs (in excess) are added to the mixture to initiate the synthesis of two new chains complementary to the original DNA chains. DNA polymerase adds nucleotides to the 3′-OH end of the primer, and strand growth extends across the target DNA, making complementary copies of the target. [Note: PCR products can be several thousand base pairs long.] At the completion of one cycle of replication, the reaction mixture is heated again to denature the DNA strands (of which there are now four). Each DNA strand binds a complementary primer, and the cycle of chain extension is repeated. By using a heat-stable DNA polymerase (for example, Taq polymerase) from a bacterium (for example, Thermus aquaticus ) that normally lives at high temperatures (a thermophilic bacterium), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle. Typically 20–30 cycles are run during this process, amplifying the DNA by a million-fold to a billion-fold. [Note: Each extension product of the primer includes a sequence complementary to the primer at the 5′-end of the target sequence. Thus, each newly synthesized polynucleotide can act as a template for the successive cycles This leads to an exponential increase in the amount of target DNA with each cycle hence the name “polymerase chain reaction.”] Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

B. Advantages of PCR The major advantages of PCR over cloning as a mechanism for amplifying a specific DNA sequence are sensitivity and speed. DNA sequences present in only trace amounts can be amplified to become the predominant sequence. PCR is so sensitive that DNA sequences present in an individual cell can be amplified and studied. Isolating and amplifying a specific DNA sequence by PCR is faster and less technically difficult than traditional cloning methods using recombinant DNA techniques. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

C. Applications PCR has become a very common tool for a large number of applications. These include: Comparison of a normal cloned gene with an uncloned mutant form of the gene: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laboriously cloning the altered DNA. Detection of low-abundance nucleic acid sequences: For example, viruses that have a long latency period, such as HIV, are difficult to detect at the early stage of infection using conventional methods. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when only a small proportion of cells is harboring the virus. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

C. Applications, contin… Forensic analysis of DNA samples: DNA fingerprinting by means of PCR has revolutionized the analysis of evidence from crime scenes. DNA isolated from a single human hair, a tiny spot of blood, or a sample of semen is sufficient to determine whether the sample comes from a specific individual. The DNA markers analyzed for such fingerprinting are most commonly short tandem repeat polymorphisms. These are very similar to the VNTRs, but are smaller in size. [Note: Verification of paternity uses the same techniques.] Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

C. Applications, contin… Prenatal diagnosis and carrier detection of cystic fibrosis: Cystic fibrosis is an autosomal recessive genetic disease resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation is a three-base deletion that results in the loss of a phenylalanine residue from the CFTR protein. Because the mutant allele is three bases shorter than the normal allele, it is possible to distinguish them from each other by the size of the PCR products obtained by amplifying that portion of the DNA. The results of such a PCR test can distinguish between homozygous normal, heterozygous (carriers), and homozygous mutant (affected) individuals Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Genetic testing for CF using PCR. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Beginning with a single piece of DNA, PCR can generate 100 billion identical copies of a specific DNA sequence in an afternoon, using a highly automated system. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

94-96ºC Region of interest Reem Sallam, MD, PhD

DP 50-65ºC UP Reem Sallam, MD, PhD

p p 72ºC Reem Sallam, MD, PhD

Exponential amplification of genes by PCR Cycle 4 Cycle 3 The gene of interest Cycle 2 Cycle 1 Cycle 36 Reem Sallam, MD, PhD 22 = 4 23 = 8 24= 16 236= 68billion

Rrecombinant DNA & recombinant proteins

Recombinant DNA Technology ( Genetic Engineering) Techniques for cutting and joining DNA Reem Sallam, MD, PhD

Reem Sallam, MD, PhD

Southern Blotting Northern Blotting Western Blotting Blotting techniques: Southern Blotting Northern Blotting Western Blotting

The Southern blotting Southern blotting is a technique that can detect mutations in DNA. It combines the use of restriction enzymes, electrophoresis, and DNA probes. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

A. Experimental procedure This method, named after its inventor, Edward Southern, involves the following steps DNA is extracted from cells, for example, a patient's leukocytes. The DNA is cleaved into many fragments using a restriction enzyme. The resulting fragments are separated on the basis of size by electrophoresis. [Note: As the large fragments move more slowly than the smaller fragments, the lengths of the fragments, usually expressed as the number of base pairs, can be calculated from comparison of the position of the band relative to standard fragments of known size.] The DNA fragments in the gel are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. If the original DNA represents the individual's entire genome, the enzymic digest contains a million or more fragments. The gene of interest is on only one (or a few if the gene itself was fragmented) of these pieces of DNA. If all the DNA segments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands. To avoid this, the next step is performed. Use a probe to identify the DNA fragments of interest. The patterns observed on Southern blot analysis depend both on: the specific restriction endonuclease and on the probe used to visualize the restriction fragments. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

B. Detection of mutations The presence of a mutation affecting a restriction site causes the pattern of bands to differ from those seen with a normal gene. For example, a change in one nucleotide may alter the nucleotide sequence so that the restriction endonuclease fails to recognize and cleave at that site Alternatively, the change in a single nucleotide may create a new cleavage site that results in new restriction fragments. A mutation may not affect a restriction site of one specific restriction enzyme, but may be revealed by using a different restriction enzyme whose recognition sequence is affected by the mutation. [Note: Most sequence differences at restriction sites represent normal variations present in the DNA, and those found in the noncoding regions are often silent and, therefore, are generally not clinically significant.] Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

4- DNA Denature, Transfer, blocking, Southern Blotting: 1- DNA extraction 2- DNA cleavage (RE) 3- DNA Electrophoresis (based on size) - + 4- DNA Denature, Transfer, blocking, 5- Hybridization e.g. with 32P-labeled probe 6- Detection Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Northern Blotting NB is very similar to SB, except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized to a specific radioactive probe. The bands obtained b autoradiography give a measure of the amount and size of particular mRNA molecules in the sample. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Western Blotting WB (also called immunoblots) are similar to SB, except that protein molecules in the sample are separated by electrophoresis & blotted (transferred) to a membrane. The probe is a labeled antibody directed against the protein (antigen) of interest  this produces a band at the location of the protein. Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33

Lecture Source: Lippincott’s Illustrated Review of Biochemistry, 4th Edition, Chapter 33: Overview Restriction Endonucleases Southern Blotting Polymerase Chain Reaction Northern Blots Western Blots