Wk 11 - Molecular Biology

DNA polymerases are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two identical DNA strands from one original DNA molecule.

A restriction enzyme is an enzyme that cuts DNA after recognizing a specific sequence of DNA. You can think of restriction enzymes as molecular scissors.

DNA ligases catalyze the formation of a phosphodiester bond between DNA single strands in the duplex form.

A cloning vector is a genome that can accept the target DNA and increase the number of copies through its own autonomous replication.

DNA Polymerase
• Can replicate DNA in a tube
• Amplifies specific region of genome
• As little DNA as from a single cell:
-plucked hair, skin scrapping, semen, touched item

Gene - a distinct sequence of nucleotides forming part of a chromosome, the order of which determines the order of monomers in a polypeptide or nucleic acid molecule which a cell (or virus) may synthesize.

A genome is the complete set of genetic information in an organism. It provides all of the information required by an organism to function.

The central dogma of molecular biology describes the two-step process, transcription and translation, by which the information in genes flows into proteins: DNA → RNA → protein. Transcription is the synthesis of an RNA copy of a segment of DNA

Introns are noncoding sections of an RNA transcript, or the DNA encoding it, that are spliced out before the RNA molecule is translated into a protein.

The sections of DNA (or RNA) that code for proteins are called exons.

A Codon is a sequence of three nucleotides which together form a unit of genetic code in a DNA or RNA molecule.

DNA Polymerase synthesizes a DNA strand and used in DNA replication while RNA Polymerase is used during transcription to synthesize the mRNA strand. In contrast with the DNA polymerase, RNA polymerases do not necessarily require the so called primer to start the process and they actually have no proofreading systems.

PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA.

PCR uses the same molecules that nature uses for copying DNA:

Two "primers", short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;

An enzyme called polymerase that moves along the segment of DNA, reading its code and assembling a copy; and
A pile of DNA building blocks that the polymerase needs to make that copy.

What is the purpose of doing a PCR (polymerase chain reaction)?

To do PCR, the original DNA that one wishes to copy need not be pure or abundant. It can be pure but it also can be a minute part of a mixture of materials. So, PCR has found widespread and innumerable uses -- to diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human evolution, clone the DNA of an Egyptian mummy, establish paternity or biological relationships, etc.. Accordingly, PCR has become an essential tool for biologists, DNA forensics labs, and many other laboratories that study genetic material.

How is PCR (polymerase chain reaction) done?

Three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture.

Each step -- denatauration (alteration of structure), annealing (joining), and extension -- takes place at a different temperature:

Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.

Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.

Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.