Molecular biology Techniques I

Molecular biology Techniques I
Dr. Eman Khairy Lecturer of Medical biochemistry & Molecular Biology

Differentiate between different methods of gene amplification techniques
Define PCR Describe steps of a PCR cycle List PCR applications in diagnosis and in forensic medicine.

DNA amplification techniques
Background In order to study genes An appreciable amount or quantity of these genes must be available by DNA amplification techniques DNA amplification techniques produce thousands or million copies of a particular gene.

DNA amplification can proceed either:
inside a test tube e.g. PCR In vitro inside a living cell molecular cloning In vivo

I. Polymerase Chain Reaction (PCR)
Definition Steps Applications RT-PCR

Polymerase Chain Reaction (PCR)
In vitro method for DNA amplification OR Amplification of a DNA segment (e.g. a gene) in a test tube.

Polymerase Chain Reaction
Polymerase: DNA polymerase DNA polymerase duplicates DNA Chain Reaction: The product of a reaction is used to amplify the same reaction Results in rapid increase in the product

Polymerase Chain Reaction (PCR)
PCR amplify the DNA segment (or sequence) of interest up to million(s) of copies in few hours. The idea of PCR is to amplify DNA content by a technique imitating DNA replication but in a test tube.

Components in PCR reaction
Target DNA Nucleotides DNA polymerase Primers

PCR steps Denaturation. Annealing Extension.
PCR occurs in repeated cycles, each cycle includes three steps: Denaturation. Annealing Extension.

This step is carried out by the enzyme helicase
1- Denaturation Like replication, PCR cycle starts by separating the two DNA strands in the double helix. In replication This step is carried out by the enzyme helicase In PCR This step is achieved by heating the DNA sample to 90-95C for 30 seconds to break hydrogen bonds pairing the 2 DNA strands.

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A primer should be provided in the test tube.
2- Annealing Replication After the two strands are separated, DNA polymerase can not initiate DNA synthesis . Needs a primer PCR A primer should be provided in the test tube.

Primers small pieces of DNA.
They are usually synthesized of 15 to 30 bases. A primer binds the DNA at a specific region. Primers are like brackets including the specific DNA sequence for a defined gene, virus, bacteria in between the brackets.

After heating the DNA double helix is separated
DNA is allowed to cool slowly to bind to the primers at 55 C and this is called annealing. After heating the DNA double helix is separated

1) A supply of the four deoxy nucleotides
3- Extension: (A,T,C,G). 1) A supply of the four deoxy nucleotides A special type of polymerase is used which is stable at high temperature and is called TAQ polymerase (Thermus aquaticus DNA polymerase). 2) DNA polymerase

PCR cycles Denaturation, annealing and extension complete one PCR cycle taking less than two minutes. At the end of a cycle, each piece of DNA in the vial has been duplicated. But the cycle can be repeated 30 or more times. Each newly synthesized DNA piece can act as a new template, so after 30 cycles, 1 million copies of a single piece of DNA can be produced! Taking into account the time it takes to change the temperature of the reaction vial, 1 million copies can be ready in about three hours.

The next cycle will begin by denaturing the new DNA strands formed in the previous cycle

55 C

1-The polymerase chain reaction is a technique that
selectively replicates RNA probes and selectively replicates DNA selectively replicates DNA probes DNA probes RNA

RT-PCR Although PCR was originally used to amplify DNA,
it is now used to amplify RNA as well, provided that RNA is first converted to a complementary DNA (cDNA) by reverse transcriptase (RT) enzyme

cDNA Is Reverse Transcribed from mRNA
mature mRNA 5’ 3’ poly A tail Reverse transcription TTTT 3’ 5’ RNA hydrolysis 3’ 5’ DNA polymerase 成 3’ CCC 5’ 5’ GGG 3’

PCR Replication in human Type of primer RNA primers
Separates the two strands of DNA Helicase Enzyme that elongates new strand of DNA DNA polymerase Temperature PCR at a very high temperature (72 degree Celcius). Products generates a full length new DNA molecule Common Items Nucleotides are needed for elongation, DNA serves as template for new strand

2-RT-PCR is a method that is used for:
forensic analysis of DNA. amplification of genomic DNA sequences. amplification of mRNA sequences. analysis of mRNA expression.

Diagnosis of diseases by detection of pathogens
PCR Applications Diagnosis of diseases by detection of pathogens Forensic testing

Diagnosis of diseases by detection of pathogens
e.g. H1N1, hepatitis B virus (HBV), hepatitis C virus (HCV) and human papilloma virus (HPV), Human immune deficiency virus (HIV). Pathogens unable to grow in culture e.g. mycobacterium T.B, where a molecule of T.B in a sputum can be detected in a few hours by PCR, instead of ordinary culture which requires several weeks. Pathogens very slowly growing in culture

Forensic testing By amplification of DNA from a dried blood spot or one hair follicle to trace the accused person for a particular crime (DNA fingerprinting).

Synthetic Oligonucleotides Complementary DNA (cDNA)
Oligonucleotides can be synthesized and used as Probes & Primers (for PCR, cDNA) Complementary DNA (cDNA) This is synthesized on a mRNA template by the enzyme reverse transcriptase (RNA-dependent DNA polymerase).

Molecular biology Techniques II
Dr. Eman Khairy Lecturer of Medical biochemistry & Molecular Biology

Recognize the mechanism of restriction endonucleases
Define molecular (DNA) cloning. Recognize cloning strategy and its importance. List types of vectors. List applications of cloning Define gene therapy.

II. Molecular (gene) cloning
Definition Restriction enzymes Vectors Steps of cloning Purposes of cloning

II. Molecular (gene) cloning:
Definition The procedure of isolating a defined DNA sequence or gene and obtaining multiple copies of it in vivo.

Basic strategy of cloning
Isolation Ligation Transfection Selection

1-Isolation of insert Initially, the DNA fragment to be cloned needs to be isolated by cutting DNA at a precise locations, using a molecular scissors called restriction endonucleases.

Restriction endonucleases
Restriction enzymes are molecular scissors that cleave double stranded DNA

Restriction endonucleases
They recognize specific DNA sequences called restriction sites and make 2 cuts one in each strand.

What kinds of bonds are broken when restriction enzymes cut?
Covalent bonds (within a single strand) Hydrogen bonds (between strands) as a result of the strands coming apart Hydrogen bond Covalent bond

Restriction enzymes are originally bacterial enzymes.
Their function is to recognize and cleave foreign DNA (virus attacking bacteria = bactriophages), So they restrict virus attack. The bacterial DNA is protected from its own restriction endonculeases by methylation.

Restriction sites Sequences of 4,6,8 bases that have been recognized by restriction enzymes Palindrome pattern i.e read from 5’3’ in one DNA strand the same as from 5’3’ in the antiparallel strand.

GAATTC G AATTC G AATTC G AATTC G AATTC EcoRI Sticky Ends
(Cohesive Ends) GAATTC G AATTC G AATTC EcoRI G AATTC G AATTC

Types of ends produced by restriction endonucleases
Blunt ends Sticky ends

Restriction endonucleases may produce
sticky end fragments or blunt end fragments

Types of ends produced by restriction endonucleases
sticky” or cohesive ends Produced from staggered cuts The resulting DNA fragments have single-stranded (ss) sequences that are complementary to each other. Blunt” ends Produced from direct cuts The resulting DNA fragments have double-stranded and therefore do not form hydrogen bonds with each other.

The selected DNA fragment is inserted into vector.
2-Ligation to a vector Definition The selected DNA fragment is inserted into vector.

Human DNA is fragmented by a restriction endonuclease
producing sticky end pieces The vector’s DNA is cleaved by the same restriction endonuclease producing similar sticky ends

A human DNA restriction fragment
anneals to the sticky ends of the split vector’s DNA

A human DNA restriction fragment anneals to the sticky ends of the
split vector’s DNA and is ligated using a ligase enzyme Recombinant DNA

The binding of isolated DNA segment to vector DNA generates a recombinant DNA ( hybrid DNA or chimeric DNA). The vector is incubated with the fragment of interest under appropriate conditions with an enzyme exhibiting DNA ligase activity. The vector (which is frequently circular) is linearised by means of restriction enzymes.

vector A molecule of DNA that will carry and join to the gene or DNA fragment to be cloned

1-What is the natural function of restriction enzymes?
Protecting bacteria by cleaving the DNA of infecting viruses. Protecting bacteria by cleaving their own DNA. Protecting bacteria by methylating their own DNA. Protecting bacteria by methylating the DNA of infecting viruses.

Essential features of vectors:
1- capable of replication inside the host cell 2- must contain a restriction site recognized by restriction endonucleases 3- must contain a marker gene, to trace the vector after insertion ( to know which host cell accepts the vector) e.g antibiotic resistance gene can be a marker.

Types of vectors: 3-Cosmids
1-Prokaryotic Plasmids 2-Viruses They are naturally occurring viruses that infect bacteria, Bacteriophage or that infect mammalian cells (e.g. retroviruses). 3-Cosmids Are artificial recombinant plasmid can insert larger DNA fragment up to 50 kb.

Prokaryotic Plasmids Bacterial cells may contain extra-chromosomal DNA called plasmids (small, circular DNA.)

Replicate independent of chromosomal DNA
Contain a restriction site recognized by restriction endonuclease Contain a marker gene, to select the bacteria containing recombinant DNA e.g antibiotic resistance. Can be readily isolated from bacterial cells and reintroduced into them. The most commonly used cloning vectors are E. coli plasmids.

They include three functional regions:
(1) an origin of replication (2) a drug-resistance gene (3) a region where DNA can be inserted without interfering with plasmid replication or expression of the drug-resistance gene.

Following ligation, recombinant DNA is transfected into host cells
3-Transfection Following ligation, recombinant DNA is transfected into host cells i.e. moving recombinant DNA from test tubes into host cell that can provide the enzymatic machinery for DNA replication.

4-Selection Selection of host cells containing the recombinant DNA by testing some markers carried by recombinant DNA as antibiotic resistance in vector.

These transformed cells, which carry the vector drug-resistance gene, can be selected by plating on nutrient agar containing the antibiotic. All the cells in each colony that grows on this medium contain vectors.

Purposes of Gene Cloning
To study genes in the laboratory, it is necessary to have many copies Hundred of human protein drugs are now being synthesized by cloning because they can be produced at a large scale and they are not immunogenic e.g insulin, erythropoietin, interferons. Some vaccines are prepared by cloning e.g hepatitis B vaccine, H1N1 (swine flu) vaccine.

PCR CLONING Gene amplification IN VITRO (TEST tube) Need less time
More efficient IN VIVO Need more time Less efficient Requirements Taq Polymerase Thermal cycler Primers nucleotides Restriction Endonucleases Ligase Vectors Host cells Application More application 1-Diagnosis of different diseases 2-Forensic tests Less application 1-study genes in lab 2-Synthesis of protein drugs 3-Preparation of vaccine.

What is Gene Therapy It is a technique for correcting defective genes that are responsible for disease development Is the most common approach The abnormal gene would be swapped by homologous recombination Would cause a return to normal function Control expression of genes. Similar to epistasis, when one gene affects the expression of another gene.

The goal of this technique is to insert a normal 'cloned' DNA for a gene into the somatic cells of a patient who has a defect in that gene.

How is it done? Viral Vector Carrying Healthy Gene
Cell with mutated gene(s) Vector inserts healthy gene into cell New gene in the cell Functional proteins are created from the therapeutic gene causing the cell to return to a normal state.

First gene therapy trial for ADA deficiency -1990

SCID and gene therapy The cloned DNA can be inserted through specific viral vector 'e.g. retrovirus'. It must permanently integrate into the patient's chromosome and expressed to produce the correct protein needed for the normal function of the affected cell.

Applications Patients with severe combined immunodeficiency disease "SCID', Some cancer patients Other diseases include: growth hormone deficiency, thalassemia, sickle cell, phenyl ketonuria, lesch-Nyhan syndrome, and Huntington's chorea.

Limitations and drawbacks
Some of the treated SCID patients developed leukemias due to activation of oncogenes.

Molecular biology Techniques I

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