Concepts in Molecular Biology

• DNA serves as the genetic material responsible for inheritance across generations.
• DNA consists of nucleic acids, which include five-carbon sugars, acidic phosphate, and four nitrogen-rich bases.
• RNA and DNA differ in their sugar composition; RNA contains ribose, while DNA contains 2-deoxyribose.
• DNA contains the bases adenine, guanine, cytosine, and thymine, whereas RNA contains uracil instead of thymine.
• Adenine always pairs with thymine, and guanine always pairs with cytosine.
• DNA carries genes influencing traits; these consist of four bases A, C, G, and T which make up codons for 21 amino acids.
• DNA is self-replicating and transcribed into mRNA for protein synthesis.
• Reverse transcription involves synthesizing DNA from RNA, where mRNAs are transcribed into complementary DNA (cDNA).

Central Dogma Qualifications

• Genes are not fixed.
• There are many transposable genetic elements in the eukaryotic genome.
• DNA sequence and protein amino acid sequence are not entirely collinear.
• Coding sequence exons are interrupted by non-coding sequence introns RNA interference occurs, where small RNA molecules help regulate gene expression.

Recombinant DNA

• Recombinant DNA is from a donor organism inserted into a vector DNA to create new DNA.
• This is also known as molecular cloning.
• Cloning reproduces daughter cells from a single cell through fission or mitosis, resulting in a genetically identical clone.

DNA Cloning

• Essential tools are restriction endonucleases, gel electrophoresis, vectors, and host cells.
• Restriction endonucleases are enzymes that cleave DNA at specific sequences, allowing scientists to cut and paste DNA in a controlled manner.
• These restriction endonucleases, isolated from bacteria, are classified into types 1, 2, and 3 based on their mechanisms of action.
• Gel electrophoresis enables isolation and purification of DNA fragments and is used to isolate and purify vectors and inserts for recombinant DNA.
• Vectors are DNA molecules that carry foreign DNA fragments into a host organism for producing large quantities of target DNA or expressing foreign genes.
• Plasmids are circular bacterial genetic elements replicating independently of the chromosome.
• Polymerase Chain Reaction (PCR) provides an alternative method for cloning and isolating large amounts of a single DNA fragment or gene.

Important Points

• Central dogma: DNA → RNA → protein.
• Proteins have structural, enzymatic, and gene regulatory functions, which translate a cell's genotype into its phenotype.
• DNA is the genetic material.
• DNA, a double helix, contains two antiparallel strands of stacked nucleotides paired through hydrogen bonds, where adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).
• DNA structure determines function. The sequence of nucleotides on one strand dictates the sequence of its complementary strand, crucial for semi-conservative replication.
• A gene is transcribed into precursor RNA, and the spliced mRNA is translated into the amino acid sequence of the coded protein; mRNA sequence unequivocally determines the protein sequence.
• Recombinant DNA is DNA from one organism inserted into a carrier vector, and when the foreign gene is introduced into a host organism, it is functional because the genetic code is universal.
• DNA cloning introduces recombinant genes from complex animals, like humans, into simple organisms, like bacteria or mice, for structural and functional studies.
• Restriction endonucleases are bacterial enzymes that recognize and cut specific DNA sequences, and are fundamental for DNA cloning.
• Gel electrophoresis separates nucleic acids by size.
• The most common host cell is E. coli bacteria.
• Plasmids, the common vectors, are independently replicating circular DNA molecules that enable resistance to antibiotics (selectable marker) and includes restriction enzyme sites for inserting recombinant genes.
• mRNA is transcribed into complementary DNA (cDNA) via reverse transcription.
• Automated fluorescent DNA Sequencing relies on the Sanger method.
• DNA sequencing of cloned cDNA for a gene is the easiest way to determine the amino acid sequence of a protein.
• Genomic and cDNA libraries are clone collections containing the genetic material of a cell.
• Base pairing between complementary DNA or RNA strands (hybridization with a labeled probe,) identifies specific nucleic acid sequences in complex mixtures.
• Southern blotting, Northern blotting, and dot blotting use hybridization for nucleic acid sequence recognition.
• Polymerase chain reaction (PCR) serves as an in-vitro method for DNA amplification.
• Molecular polymorphism is studied at the genetic level by DNA typing methods relevant for transfusion medicine which include restriction fragment length polymorphism, allele-specific oligonucleotide probe hybridization, allele-specific PCR amplification, DNA sequencing, and DNA profiling (DNA fingerprinting).
• PCR used for early detection of transfusion-transmitted pathogens.
• Gene therapy and the usage of recombinant proteins like interferons are other therapeutic applications of molecular biology, coagulation factors, & growth factors.
• Molecular RBC antigen typing confirms serologic testing or resolves inconsistencies.

Review Questions

• The central dogma says DNA is the genetic material.
• Recombinant-DNA technology is possible because the genetic code is universal.
• Agarose gel electrophoresis separates DNA molecules by size.
• Restriction fragment length polymorphism (RFLP) is related to use of bacterial endonucleases.
• Polymerase chain reaction (PCR) amplifies DNA.
• Plasmids are vectors used for molecular cloning.
• Some model organisms simplify human disease study, are used to produce recombinant proteins, and include prokaryotes and some eukaryotes.
• DNA sequencing is an enzymatic in-vitro reaction.
• RFLP (restriction fragment length polymorphism) and SSP (sequence specific primers) are techniques used for DNA typing.
• Recombinant DNA techniques are useful research tools.
• Transcription mediated amplification requires thermostable DNA polymerase, is an isothermal procedure, and utilizes probes labeled with fluorescent tags.
• The preseroconversion window is the time when an infected person may not test positive via serologic methods, which may be shortened using molecular techniques, and refers to viral pathogens.
• Red blood cell molecular antigen typing is useful except in quantitative gene expression analysis.