PMLS-1: ADVENT OF MOLECULAR DIAGNOSTIC TOOLS

Molecular diagnostics

Biochemistry + molecular biology

Molecular biology

where replication, translation, and transcription comes in

Biochemistry

nucleic acids, DNA, RNA, and their composition (nitrogenous bases)

Molecular diagnostics

• A branch of clinical diagnostics that utilizes genetic material (DNA or RNA) in disease diagnosis

• Combination of biochemistry and molecular biology applied for diagnostic testing particularly covering three major clinical areas: infectious diseases, genetics, and tumor markers

• Infectious disease

• Genetics

• Tumor Markers

Molecular-based diagnostic tests ⟶ 3 Major Clinical areas

sickle cell anemia

was categorized as a molecular disease in 1949

Phosphate synthesis

an assay developed for small oligodeoxynucleotides

 Arthur Kornberg

who isolated DNA polymerase

Roy Britten

who made the initial hybridization methods and electrochemical DNA detection

Solid-phase oligodeoxynucleotide synthesis and enzymatic synthesis

two synthesis for short RNAs

Gall and Pardue

who developed In situ hybridization technique

 in place

what does in situ mean?

Isolation of the first restriction enzyme and reverse transcriptase

What did Hamilton Smith develop

Restriction enzyme

synonymous to scissors because it cuts at certain sites

Reverse transcriptase

Known because it is one of the characteristics of HIV

Nucleic acid hybridization methods

what methods was developed during 1970

to combine

what does hybridization mean?

Hybridization techniques

utilize the complementarity of the bases

Southern blotting technique

what technique was developed in 1975

First Generation Sequencing technique - Sanger sequencing

what sequencing was developed during 1977

Fredrick Sanger

who developed the first sequencing technique

Sanger sequencing

also known as dideoxy sequencing or chain termination sequencing because as it gets longer, it is cut, and then the marker lights up

Maxam-Gilbert Sequencing Method

what was the sequencing method developed in 1980

Allan Maxam and Walter Gilbert

who developed the sequencing method

Maxam-Gilbert Sequencing Method

also known as chemical sequencing because it uses chemicals to degrade the structure and treat afterwards to visualize the whole sequence

Restriction Fragment Length Polymorphism (RFLP) analysis

what analysis was established in 1985

Polymorphism

your minute differences from everyone else and uniquely in your structure or genome

RFLP

The principle of ___ is utilized in forensics, specifically in fingerprinting to compare samples and find which match because whatever cuts in the sample at which site, when compared to others, it will also cut at the same site, so when you visualize it, they look the same

In the ___, you utilize restriction enzymes that cut at certain sites, so it can recognize where to cut

Polymerase Chain Reaction

what was invented by Kary Mullin in 1985

Polymerase chain reaction

mimics the DNA replication process

Fluorescent In situ Hybridization (FISH)

developed in 1986.

Mostly used, particularly in tissues, right now, so there will be markers in the chromosome and that is where the fluorescent label will attach and if there is the presence of that certain genome where it attached, it will light up

 thermus aquaticus

where did they discover the first thermostable DNA polymerase

first DNA chip conceptions

an invention from 1988-1991 that allows you to analyze multiple samples per dot

DNA/RNA mimics

designed in 1991, utilized for PCR, so that its building blocks will be better, particularly the peptide nucleic acids

qPCR (quantitative PCR)

conception of ___ in 1992

PCR

its purpose is to amplify (padamihin) the sample since it mimics the replication process of the DNA

whole genome amplification

and Strand-displacement amplification

which amplifications were assays created for in 1992

Comparative genomic hybridization (CGH)

allows the checking of any conformational changes with your sample without necessarily performing any cell culture, so you test directly

endonucleases

what was discovered for invasive cleavage assays

DNA topological labelinginven

an invention in 1994.

they mark at the chromosome, so they can map where the area is and, usually, they utilize fluorescence

rolling amplification

invention is 1995 that ispart of circular probes

 DNA microarrays

1996 was the first application of __
A glass slide could house multiple samples and that is per dot

Pyrosequencing technique

The next generation sequencing (2nd generation)

Lab-on-a-ChiP (microfluidics)

created in 1998 for DNA analysis

ELISA

Western Blot

Immunostaining

what immunoassays were developed in 1985-1999

 Massively parallel sequencing (MPS)

what did Lynx Theraputics develope in the year 2000

protein profiling assays

what did they apply in the diagnosis of human disease in 2001

Pacific Biosciences

who invented the Single molecule real time sequencing(SMART)

George M. Church

who invented Polony sequencing

CRISPR

clustered regularly interspaced short palindromic repeats (that is what they’re looking for in the section of the DNA)

Emmanuelle Charpentier and Jennifer Doudna

who developed the gene editing tool, CRISPR

Chromosomes

• contain the genetic material

• are tightly coiled and reveal the histones and nucleosomes which are tightly packed (when uncoiled even further, you would have the DNA)

double helix

Structure of the DNA

Rosalind Franklin

What is (1)

The one who visualized the DNA is (1)__ and that radiograph picture is called a photo 51 (she was only recently credited because she is a girl)

Photo 51

What is (2)

The one who visualized the DNA is (1)__ and that radiograph picture is called a (2)__ (she was only recently credited because she is a girl)

Haploid

Chromosomes can be described either as:

• __ - 23 (only one set of copies)

Diploid

Chromosomes can be described either as:

• __ - 46

DNA

1. Usually double- stranded

2. Thymine as a base (where adenine binds)

3. (Both utilize pentose or sugar containing five carbon atoms) Deoxyribose as the sugar (deoxy meaning no oxygen usually in the second carbon)

4. Maintains protein- encoding information (that is transcribed to RNA)

5. Cannot function as an enzyme

6. Persists (more stable, not delicate, allows bench-stop work and stronger because of the double-  stranded nature)

RNA

1. Usually single- stranded

2. Uracil as a base (where adenine binds)

3. (Both utilize pentose or sugar containing five carbon atoms) Ribose as the sugar 

4. Carries protein- encoding information (which facilitates the translation into proteins) and controls how information is used

5. Can function as an enzyme

6. Short-lived (everything has to be cold, clean, and you need an ice bucket in the area)

Purines

double carbon nitrogen rings and under it is adenine and guanine

Pyrimidines

single carbon nitrogen rings and under it is cytosine, thymine, and uracil

Nucleotide

the structure is phosphate, sugar, and nitrogenous base

Nucleoside

if only the sugar and the base are present (if there is no phosphate)

 CG or GC islands

Strongest binding of nitrogenous bases.

They are strong because they contain three hydrogen bonds, so it’s harder to separate them

rRNA

TYPES OF RNA:

• So that will meet and the ribosome has two units (small and big) that will encapsulate or sandwich the mRNA in between

mRNA

TYPES OF RNA:

• The DNA is copied (one strand) by the RNA polymerase to make the mRNA because what is copied is the list of instructions and when the rRNA and mRNA are sandwiched, that will attract tRNA

tRNA

TYPES OF RNA:

• contains the anticodon (codon means three or every three bases) or the opposite (example: if the mRNA has UAA, the tRNA has AUU) because it needs to hybridize or attach and the tRNA carries amino acids (methionine, valine, lysine, etc. and the start codon is fMet or methionine) that will attach with each other and that chain of amino acids is now a polypeptide and since it is one straight chain, it is a primary sequence that is a type of protein (polypeptide - many peptide bonds)

mRNA

size: 500 - 4500+

function: Encodes amino acid sequence

rRNA

size: 100 - 3000

function: Associates with proteins to form ribosomes which structurally support and catalyze protein synthesis

tRNA

size: 75 - 80

function: Transports specific amino acids to the ribosome for protein synthesis

Replication

generating or producing new DNA from parent DNA

Semiconservative replication

• original DNA is conserved (1 newly synthesized strand/daughter strand + strand from the original DNA)

• Helicase, topoisomerase, gyrase, etc. will unwind or open the DNA (if not, it can’t pass through since it is tightly coiled) to separate them and DNA polymerase will work to create a new strand and that new strand will contain one daughter strand and a strand from the original DNA (that’s why it is semiconservative) 

• DNA replicates itself to produce DNA

• DNA is transcribed to make RNA (transcription)

• RNA is translated to produce protein (mRNA is translated into polypeptide)

Central Dogma (3)

Transcription

 genetic information from the sequence of bases in DNA is transcribed into RNA (specifically mRNA), facilitated by the RNA polymerase

Translation

• base sequence in the RNA determines the amino acid sequence of the protein that is synthesized  (using the base sequence of the mRNA, it will use that to make amino acids sequence or polypeptide chain) 

• The instructions are there and it is read which is facilitated by the rRNA (where it will dock or place) and tRNA (supplies the anticodon with the alternative amino acid that will attach with the peptide chain)

Nucleic Acid Isolation

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• The process of separating DNA or RNA from its surrounding material

• In nucleic acid isolation, blood is centrifuged to get the particular component that you need which is the white blood cells (you isolate the part that contains a nucleus)

• The difference when you isolate DNA and RNA is if you are to isolate RNA, it is more sensitive because it is labile (easily breaks or degrades)

Nucleic Acid Quantification

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Determination of the average concentration of DNA or RNA present in a sample

Nanodrop

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Nucleic Acid Quantification types

  • __ - most common because you only need to deposit or pipette a small amount of around 0.2 µL at the spot and it utilizes spectrophotometric analysis

OD (optical density)

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Nucleic Acid Quantification types

  • __ -  spectral reading which is read for the concentration

Qubit (under Thermo Fisher)

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Nucleic Acid Quantification types

  • __ - fluorogenic which utilizes fluorescence

Quantitative PCR or real time PCR

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Nucleic Acid Quantification types

  • __ - you can see the increase of the copies of the sample in real time (fluorogenic in nature or otherwise utilizes a probe)

Polymerase Chain Reaction

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Used to amplify target DNA sequence

Target

Probe

Signal

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Polymerase Chain Reaction types (3)

Reverse Transcriptase PCR

• utilized when the sample is RNA because it cannot undergo the three steps of the polymerase chain reaction

Denaturation

PCR STEPS:

• separation of the strands through increased temperature, usually at around 90-95° C, causing the DNA to unwind

Annealing

PCR STEPS:

•  process of attachment or hybridization of primers

Primers

short RNA sequence and its purpose is to facilitate or guide where the DNA polymerase will attach

Extension

PCR STEPS:

•   formation of the new strand which is facilitated by the DNA polymerase enzyme

DNA polymerase

DNA replication in the human being

Taq polymerase

• is an example of the DNA polymerase extracted from the Thermus aquaticus bacteria

• is used in vitro or in the PCR itself (PCR is in vitro)

RT-PCR

converts RNA material into DNA first before it is amplified

Multiplex PCR

• most famous nowadays because during the pandemic

• facilitated running of respiratory samples or screening of respiratory infections

BioFire and bioMérieux

example brands of multiplex PCR

Expression cloning

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Copying of DNA that codes for a protein of interest in a plasmid (“expression vector”)

• Facilitates the technique of the recombinant technology

recombinant technology

In the __ ,we are utilizing a target genome or gene to be incorporated into a vector, typically a plasmid (found in a bacterial cell that is extrachromosomal meaning it is just something extra that is not needed for sustenance of life) which facilitates antibiotic resistance or virulence factors in bacteria

• This is the target gene or gene of interest for insulin

• It is attached to the plasmid

• The plasmid is cut, facilitated by the restriction enzymes

• After cut, it is attached or ligated to the plasmid

• Once completed, the plasmid is inserted again into a bacteria for expression cloning to produce the desired compound

Steps of Expression Cloning for insulin (5)

insulin and hepatitis B vaccine

• The most well known in recombinant technology is the production of __

Gel electrophoresis

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Separate DNA, RNA, and proteins by means of an electric field

• The electricity will pass through the gel

• Used to estimate the size of DNA following PCR

Voltage

LABORATORY TECHNIQUES IN MOLECULAR DIAGNOSTICS:

• Gel electrophoresis

  • __ is applied to both ends so that electricity can pass through it, then it will proceed depending on the nature of the fragments, whether they are short or long, and its polarity.