Chapter 04 Concepts in Molecular Biology

• Molecular biology is the study of molecular interactions in living cells, focusing on nucleic acids like DNA and RNA.
• According to the central dogma, genetic information flows from DNA to RNA to proteins.
• Gene expression involves transcription and translation, leading to unique protein molecules that determine cell structure and function.

DNA Is the Genetic Material

• Living organisms replicate and pass on hereditary traits or genes to their offspring.
• In 1928, Frederick Griffith's experiment with Streptococcus pneumoniae showed DNA carries genetic information.
• Virulent S strain kills mice, while nonvirulent R strain doesn't, heat-killed S strain doesn't cause disease, but a combination of heat-killed S strain and live R strain is lethal, transforming the R strain, this is known as Griffith's transformation.
• Griffith's experiment is the basis for genetic material transfer from one organism to another in labs.
• In 1944, Oswald T. Avery's group purified the "transforming principle" and found it consists mainly of DNA, DNase treatments affect DNA and cause loss of activity.
• In 1952, Alfred Hershey and Martha Chase's experiment with bacteriophages confirmed that genes are made up of DNA.
• Bacteriophages with radioactive sulfur (35S) or phosphorus (32P) were used to infect bacteria. The 32P DNA in pellet lead to a new generation of phages by centrifugation.

Features Relevant to Molecular Techniques

• In 1950, Erwin Chargaff's rules from DNA quantitative analysis showed the discovery to the structure
• In 1953, James Watson and Francis Crick proposed that DNA is a double helix.
• The DNA double helix is built of deoxyribose and phosphate, with paired nitrogen nucleotides held by hydrogen bonds.
• Adenine (A) pairs with thymine (T) with two hydrogen bonds, while guanine (G) pairs with cytosine (C) with three hydrogen bonds.
• Polarity labels 3' and 5' determine the direction of DNA replication and transcription.
• Complementarity resulting from hydrogen bonds is key to DNA strand separation (denaturation) and rejoining (renaturation).
• The DNA melting point (Tm) is the temperature at which half of the hydrogen bonds are broken.
• Temperatures above Tm promote strand separation, while temperatures below Tm keep strands together.
• The Watson-Crick model explains DNA replication, in which strands separate and act as templates for complementary halves. Each daughter DNA contains an "old" and a "new" strand, making replication semiconservative.
• In eukaryotes, DNA polymerase III forms new strands from deoxyribonucleoside triphosphates (dNTPs) with double-stranded DNA and magnesium.
• DNA sequencing and polymerase chain reaction (PCR) mimic DNA replication.

Expression of Genetic Information

• Genes determine the sequence of amino acids and structure of proteins.
• The molecular basis of sickle cell anemia showed that DNA nucleotide order determines the amino acid sequence of a protein.
• Sickle cell anemia is an autosomal-recessive genetic disorder that is due to the B-globin chain on chromosome 11.
• Substituting valine for glutamic acid at the sixth amino acid position of what causes defective hemoglobin with patients.
• Proteins are synthesized in the cytoplasm, with ribosomes being the major component for protein synthesis.
• Messenger RNAs (mRNAs) are RNA molecules that convey genetic information from the nucleus to the cytoplasm
• Transfer RNA (tRNA) molecules have amino acids attached and align with mRNA codons via anticodon sequences that provide each tRNA for specificity.
• Marshall Nirenberg and Har Gobind deciphered that mRNA codons correspond to specific amino acids. Degeneracy of genetic code protects it from devastating mutation effects in the third or second base of a triplet.

The Central Dogma of Molecular Biology, Expanded

• The classic theory confirmed that is DNA is self-replicating and transcribed into mRNA to create proteins.
• RNA viruses replicate via DNA synthesis in host cells with retroviruses replicate via the synthesis of a DNA provirus, which means DNA can be synthesized from RNA.
• CDNA is derived from exons
• Genes can behave as genetic elements inside the genome and can participate in DNA recombination Coding sequences are interrupted by introns

Recombinant DNA

• Fragmented DNA combined with vector DNA is introduced to a simpler host for replication
• It is called molecular cloning

The Coding Sequence of a Gene

• The coding part of genes are made out of exons and introns.
• Newly transcribed RNA (pre-mRNA) becomes heterogenous RNA (hnRNA) and loses introns in the process of splicing
• Both genomic DNA and the coding sequence are cloned
• The mRNA is transcribed in vitro to its cDNA using synthetic or retroviral enzymes

Tools for DNA Cloning

• The essential tools that help define the specificities of molecular cloning involve restriction endonucleases, gel electrophoresis, vectors, etc
• Enzymes like restriction endonucleases cut DNA at certain sequences that allow scientists to "cut and paste"

Restriction Endonucleases

• These are a result and cause of the isolation of certain bacteria
• They are classified as type I, II, and III by their mechanism of function.
• They usually recognize sequences 4 to 8 nucleotides in length
• Recognition sequences are palindromic
• The name given to each enzyme reflects its origin
• They can generate blunt ends that cut both strands in the middle
• Enzymes like EcoRI recognize sequences GAATTC then cuts after the first G in both strands, leaving unique ends

Gel Electrophoresis

• It is a technique to examine restriction patterns
• This allows visual analysis of DNA length fragments
• DNA samples are loaded into a gel immersed in buffer
• DNA strands move towards an anode (+) as DNA is negatively charged
• Agarose gels are produced and a plastic comb is placed before it cools to a gelatin state.
• Wells in the cell contain DNA fragments
• DNA run goes inversely proportional to molecular weight

Vectors

• Vectors transport foreign DNA into a host cell DNA through a known nucleotide sequence
• This allows for larger quantities of protein production as long as a vector is present.
• Plasmids are the simplest kind of vectors that are made of bacterial circular genetic elements These designer plasmids are modified to be perfect DNA carriers.
• All plasmids contain an origin of replication.
• They can contain genes that encode for antibiotic resistance.

Plasmid Isolation

• The process in which plasmids are separated from a host's chromosomal DNA due to their small size + circular structure
• This creates a small preparation of purified plasmid The procedure involves overnight culture gets centrifuged. treatment with a solution of SDS and NaOH, causing all cell contents to release. Using a solution of potassium acetate and acetic acid neutralizes the solution and causes everything else to fall into a precipitate.

Recombinant DNA Libraries

• Production of recombinant libraries is fishing for a gene out of a source genome
• Target genes are extracted by way of a vector in the DNA
• Clones and genome and mRNA are selected by screening with labeled cDNA

Expression of Cloned Genes: Recombinant Proteins in Clinical Use

• Using engineered in-vitro recombinant proteins has improved treatment for several diseases due to the high cost
• Cloned genes are inserted into expression vectors that direct the production of recombinant proteins
• A constantly increasing list of therapeutic agents are including Interferon-a to treat hairy cell leukemia, recombinant hepatitis B vaccines, recombinant coagulation factor IX etc

The Polymerase Chain Reaction

• PCR isolates large amounts of DNA for study in vitro under defined segments
• The DNA is replicated by synthetic reagents and and cyclic changes to the reaction temperature
• Synthetic oligonucleotides serve as the forwards + reversed
• PCR has many important applications
• The number of molecules can continue to grow indefinitely with a theoretical yield for 1BN progeny molecules for each molecule created. However, the accumulation of a product plateaus depending on target reagents

DNA Sequencing

• Individual DNA is obtained for nucleotide sequencing by molecular cloning and PCR
• The protein is determined by its coding for its amino acod sequencing using the method conceived at Medical Research Council's Lab in Cambridge by Fred Sanger
• The Sanger sequencing is an in-vitro process mimicking DNA synthesis
• It can detect homologous DNA or RNA sequences

Southern Blotting

• Southern blot digest the DNA to isolate the ones needed through agarose gel electrophoresis with stained bands
• The nylon membrane isolates a single sequence by a labeled probe.
• This labor intensive procedure is often used to replace new PCR detection methods

Northern Blotting

• It is a process similar to Southern blotting, but RNA instead is placed.
• The method uses total RNAs then fractioned in size through gel electrophoresis

DNA Microarrays

• Also called gene chips, allow tens of thousands of genes analyzed at once
• Common applications include tumor vs normal cells
• Uses blood from Illumina BeadChip microarray

Fluorescent In Situ Hybridization

• The use of fluorescent probes to find what cells or chromosomes are homologous to
• Probes are used in subcellular level
• Was originally conceptualized to localize the ABO gene in 1995.
• DNA is amplified, with more than 100k copies detected, through use of the PCR technique

Real-Time PCR

• Product formed during each cycle of amplification is measured by fluorescence at the rate and pace in which its produced
• Is very advantageous because it determines how many samples can come forth

Reverser Transcriptase PCR

• Copies of RNA are adding by a CDNA through reverse transcription before the PCR amplification cycle happens
• Is used for high sensitivity detection of DNA and RNA
• FDA recently approved it for use in testing donors' blood

Transcription Mediated Amplification

• TMA starts by binding to RNA, which is an enzyme responsible for RNA synthesis
• Light is captured and emitted as RNA from probes
• NAT helps detect pathogens through this process, including HIV and hepatitis B and C
• Has reduced testing time through the ability to detect multiple pathogens concurrently.

Antibodies as Probes for Proteins

• Is used on mRNA, also used on labelled antibodies
• Also used for Western Blotting which uses polyacrylamide to separate based on sizes

Techniques for Studying Gene Polymorphism

• RHLP determine is a person is A0 from either BO
• In the sixth position on the defected genes is the same codon on the normal
• Has specific PCR probes
• A commonality comes with genotyping as certain levels.

DNA Sequencing

• Allelles that are polymorphic can specifically be amplified and sequenced in plasmid vectors

HLA Typing for Allogeneic hematopoietic stem cell transplant (HSCT) and DNA Profiling (“Fingerprinting”)

• Minisatelites are parts of the human genome with variable nucleotide length
• Microsatallites contain 2-5 repeated units
• These parts are highly polymorphic
• FBI has made it a standard to include 13 STRs

Systems Biology and Red Cell Genotyping

• Moderns system still are a valuable tool in nucleic acids in cellular process.
• Systems that high genomic output
• Integration from in vitro all work that goes into silico
• Molecular typing of blood in those that we test makes for easier tests.

Genetic Basis of Blood Groups

• Cloning had early on efforts
• Most were coding one gene and four exceptions
• DNA testing and has been great

Clinical Application of Red Cell Genotyping

• DNA Is not sent fast enough or in an exact moment, has to follow an established and routine test
• It helps to understand how someone of the zygosity, whether heterozygous or homozygous, is.
• Fetal typing or maternal samples is not necessarily accurate

Blood group typing

• DNA typing can be used and or provide the most accurate results than not The costs that are had are less accepted in total.