Biochemical techniques used in molecular genetics

overviewRecombinant DNA technologyRestriction endonucleasesVectorsDNA cloningPolymerase chain reactionBlotting techniques

Recombinant DNA technology1. Recombinant DNA technology is genetic

engineering which effects artificial modification of the genetic constitution of a living cell by introduction of foreign DNA through experimental techniques.

Tools of Recombinant DNA technology.

Restriction endonucleases

Cloning of DNA

Probes

ENZYMES Restriction endonucleases Exonucleases Endonucleases Reverse transcriptase DNA polymerases DNA ligase

palindrome

Restriction endonucleasesSpecial group of bacterial enzymes which cleave

double stranded DNA into smaller more manageable fragments.

Restriction endonucleases cut the DNA at the palindome , which is short stretch of DNA (4-6 bp) which exhibit two fold symmetry.

A restriction enzyme is named according to the organism from which it was isolated. Hae111

Sticky and blunt ends

Restriction site is the DNA sequence recognized by a resrictriction enzyme.

vectors

It’s a molecule of DNA to which the fragment of DNA to be cloned is joined. It’s a molecule of DNA to which the fragment of DNA to be cloned is joined.

Types of vectors1. Bacterial plasmids2. Bacteriophage lambda3. Cosmids4. Retroviruses

Essential properties of a vector:

It must be capable of autonomous replication within a host cell.

It must contain at least one specific nucleotide sequence recognized by a restriction endonuclease.

it must carry at least one gene that confers the ability to select for the vector, such as an antibiotic resistance gene.

plasmids:Prokaryotic organisms contain single, large,

circular chromosome.

In addition, most species of bacteria also normally contain small, circular, extra-chromosomal DNA molecules called plasmids.

Plasmid DNA undergoes replication that may or may not be synchronized to chromosomal division.

Plasmids may carry genes that convey antibiotic resistance to the host bacterium, and may facilitate the transfer of genetic information from one bacterium to another.

The plasmids are the most commonly used vectors and can accept short DNA pieces about 6 to 10 kb long.

DNA CLONING

A clone is a large population of identical molecules, bacteria or cells that arise from a common ancestor.

Introduction of a foreign DNA molecule into a replicating cell permits the amplification (that is, production of many copies) of the DNA.

Steps of cloningTo clone a nucleotide sequence of interest, the

total cellular DNA is first cleaved with a specific restriction enzyme, creating hundreds of thousands of fragments.

Each of the resulting DNA fragment is joined to a DNA vector molecule (known as a cloning vector) to form a hybrid molecule

Each hybrid recombinant DNA molecule conveys its inserted DNA fragment into a single host cell, for example, a bacterium, where it is replicated (or "amplified").

As the host cell multiplies, it forms a clone in which every bacterium carries copies of the same inserted DNA fragment, hence, the name "cloning."

The cloned DNA is eventually released from its vector by cleavage (using the restriction endonuclease) and is isolated. By this mechanism, many identical copies of the DNA of interest can be produced.

recapRecombinant DNA technologyRestriction endonucleasesVectors (plasmids)ProbesDNA cloning

probesA single stranded piece of DNA labelled with a

radioisotope or antibiotic such as biotin.

The nucleotide sequence of the probe is complementary to the gene of interest, called the target DNA.

Probes therefore identify which band on gel contains the target DNA.

Polymerase chain reactionThe polymerase chain reaction (PCR) is an

enzymatic (test tube, in vitro) 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.

PCR uses DNA polymerase to repetitively amplify targeted portions of DNA.

Each cycle of amplification doubles the amount of DNA in the sample.

leads to an exponential increase in DNA with repeated cycles of amplification.

Steps of PCR1. 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 to 35 nucleotides long, which are complementary to the respective flanking sequences. These serve as primers.

2. Denature the DNA: The DNA to be amplified is heated to separate

the double stranded target DNA into single ‑strands.

3. Annealing of primers to single stranded DNA: ‑ The separated strands are cooled and allowed to

anneal to the two primers (one for each strand).

4. Chain extension: DNA polymerase and deoxyribonucleoside

triphosphates (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' hydroxyl end of the primer, and strand growth ‑extends across the target DNA, making complementary copies of the target.

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 from a ‑bacterium that normally lives at high temperatures (thermophilus aquaticum), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle.

Typically twenty to thirty cycles are run during this process, amplifying the DNA by a million fold to a billion fold. ‑ ‑

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

APPLICATIONS OF PCRComparison of a normal gene with an mutant

form of the gene for detection of mutations.

PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laboriously cloning the altered DNA.

2. Detection of low—abundance nucleic acid sequencesFor 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 cell’s is harboring (shelter) the virus.

3. 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.

4 verification of paternityUtilizes the same technique of DNA fingerprinting .

4. Prenatal diagnosis and carrier detection cystic fibrosis:Cystic fibrosis is an autosomal recessive genetic

disease resulting from mutations in the cystic fibrosis trans-membrane regulator (CFTR) gene.

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.

Uses of recombinant DNA technologyDiagnostic purpose as in PCR.Treatment purpose vaccines hormones factor 8 , TPA (tissue plasminogen activator).Genetic counsellingGene therapy (SCID).Transgenic animals

Southern blotting:Southern blotting is a technique that can detect

mutations in DNA.

Experimental procedure: This method, named after its inventor, Edward Southern, involves the following steps.

StepsFirst, DNA is extracted from cells, e.g., a patient's

leukocytes.

The DNA is cleaved into many fragments using a restriction enzyme.

The resulting DNA fragments are separated on the basis of size by electrophoresis.

The DNA fragments in the gel are denatured into single strands and transferred to a nitrocellulose membrane for analysis.

A radioisotope labelled probe is used to identify the gene of interest.

Northern blots:Northern blots are very similar to Southern

blots, 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 radioactive probe. The bands obtained by autoradiography give a measure of the amount and size of particular mRNA molecules in the sample.

Western blots:Western blots (also called immunoblots) are

similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted to a membrane. The probe is a labeled antibody, which produces a band at the location of its antigen.