Molecular Methods and Human Genome

Learning Objectives

• Overview of molecular methods in human molecular pathology
• Modern molecular approaches enhance understanding of molecular genetics
• Familiarization with common methods in molecular research and teaching labs
• Types of molecular methods
• Examples of applications for each method
• Knowledge application and integration

Importance of Molecular Methods

• They aid in understanding genes and their functions
• Clinical genetics benefit through improved diagnosis
• Allow preventative treatments
• Advances in disease treatment are made possible

Organization of the Human Genome

• Genome comprises genes, regulatory sequences, and DNA information
• The genome is divided into a large nuclear and small mitochondrial genome
• 5% of the nuclear genome is highly conserved, indicating functional importance
• Only 1.1% of the genome is protein-coding; the rest is non-coding

More on Organization of the Human Genome

• Protein-coding genes may form families through gene duplication
• Non-functional gene-related sequences (pseudogenes) can be present
• Non-protein-coding sections of the genome are functionally significant
• Non-coding RNA plays a crucial role in regulating protein coding genes

DNA Analysis Methods

• DNA can be selectively amplified through PCR and cloning
• PCR and cloning allows a sufficient quantity of a DNA sequence to be analyzed
• PCR can produce millions of copies of a specific DNA sequence in about 2 hours

DNA Amplification via PCR

• PCR is a cyclical process
• PCR works by heating and cooling DNA
• PCR helps to denature, anneal and enzymatically amplify DNA

PCR Applications

• PCR used in place of cloning (cutting up DNA and inserting it into a bacterial plasmid)
• PCR can amplify specific sequences from small amounts of material
• PCR can selectively detect DNA sequences not normally present in tested tissue (e.g. viruses)
• PCR assists analysis of degraded DNA samples

DNA Separation by Gel Electrophoresis

• Electrophoresis allows DNA separation by size and conformation
• Polyacrylamide gels are used for single-stranded DNA molecules under 500 nucleotides
• Agarose gels are more porous, suited for molecules 300-20,000 nucleotides
• Pulsed-field gel electrophoresis helps with long DNA molecules

DNA Analysis by Agarose Gel

• This is carried out by quantitation and sizing
• Samples are stained with ethidium bromide
• The resultant gel is exposed to UV light for visualization

Gene Deletion Example

• A gel image can show a gene deletion
• The gel would contain results showing the mother, father, patient, sister, pos and neg controls and bp ladders

DNA Sequencing

• DNA sequencing is used to "read" nucleotides
• Oligonucleotide primer for DNA polymerase is used
• A single-stranded DNA molecule is sequenced

DNA sequencing - cont.

• PCR, fluorescent, chain-terminating dideoxynucleotide triphosphates are needed
• Capillary electrophoresis is used with a laser
• A detector is used

Electropherogram

• Electropherograms can be used to display the sequence of a deleted gene
• The sequence of the deleted gene in the patient is compared to normal gene sequence

Genomics

• Uses DNA sequencing and bioinformatics
• Allows the understanding of structure and function of the genome
• The first genome sequenced was the mitochondrial genome in 1981
• The draft human genome sequence with 3.23 Million bases was published in 2001, costing $3 billion and over 10 years to complete
• The 1000 genomes project from 2008-2012 sequenced 2,577 individuals of multiple races -Allowed the identification and database genetic variation/polymorphisms between individuals

Improvements in genome sequencing

• There has been major technological advancements in genome sequencing
• The advancements have increased the number of kilobases read per machine

Next Generation Sequencing

• Massively parallel DNA sequencing platforms available
• High throughput that parallelizes sequencing, producing millions of sequences at once
• It has reduced DNA sequencing costs significantly
• A massive amount of sequencing data is produced (=BIG DATA)

Next Gen Sequencing hardware

• ABI SOLID (2006...gone)
• Roche 454 FLX (2007-2013)
• Illumina Genome analyser (2010-2014)
• Ion Proton (2012-current), 1 genome takes 4 hours
• Illumina HiSeq 2500 (2012-current), 1800 genomes annually or 6 genomes in 27 hours
• Oxford Nanopore MinION

RNA Analysis

• The pathway is DNA -> RNA -> protein
• RNA reading allows counting RNA amount (gene expression)
• RNA reading allows finding mutations
• RNA reading can show a new spliced mRNA that makes a "bad" protein
• RNA reading helps testing mutations that affect alternative exon splicing of the mRNA

RT-PCR

• Reverse transcriptase-polymerase chain reaction (RT-PCR)
• It analyzes individual gene transcripts/gene ‘expression’ (RNA)
• Highly sensitive, cost effective, and quick

Real time RT-PCR

• Used to assess individual RNA 'gene expression' or abundance in a sample
• The accumulation of amplified DNA is monitored continuously during PCR cycling
• This is done via changes in fluorescence
• It all happens in a single step of amplification and detection

TaqMan Probes

• Taqman probes bind to a gene target in a sequence-specific way
• When the primer pair is elongated by PCR, Taq polymerase cleaves the fluorophore using 5’-3’ exonuclease activity
• Displacement away from the quencher allows the fluorophore to glow
• A laser excites this during real-time PCR

Principal of Quantitative Real Time PCR

• Reactions are characterized by the PCR cycle where target amplification is first detected (threshold cycle ‘Ct’)
• Reactions are classified by their threshold cycles
• Increased quantity of target DNA yields a faster fluorescence increase
• This increase yields a lower Ct
• Ct values determines ‘gene expression’ differences between samples

Methods Overview

• Selective amplification: PCR and cloning allow enough DNA or RNA to be analyzed
• Specific detection: Molecular hybridization, Southern blot, microarray and libraries identify the sequence of interest

Hybridization

• It is a double-stranded DNA formation
• It occurs between two single strands that feature complementary sequences
• Involves mixing DNA from two sources denatured by heat or alkali
• Labeled DNA can identify specific DNA sequences

Principles of Hybridization

• Denaturing via heat or alkali
• Renaturing/hybridising occurs once denatured

Hybridisation Methods

• Dot blot
• Southern blot
• Fluorescence in situ hybridisation (FISH)
• Single Nucleotide Polymorphism (SNP) arrays
• Microarray
• Array Comparative Genomic Hybridisation (aCGH)

Hybridization: Expression Microarray

• Simultaneously analyzes expression profiles of thousands of genes (RNA abundance)
• Cluster of complementary DNA sequences (probes) within each dot is known
• Fluorescent dye labels and washes sample cDNA over the array
• Complementary DNAs bind, and are excited via a laser
• Spot brightness indicates cDNA presence and gene expression

Single Nucleotide Polymorphisms (SNPs)

• Approximately 10 million SNPs in the human genome (1/300nt)
• The HapMap project looks for patterns of common genetic variations, but is retired
• The HapMap is a stepping stone for the 1000 genomes project
• Databases like GnomAD built from >125k people
• Common genome variation patterns can be described
• SNPs mark locations to help search for genes relating to diseases and other things

GWAS

• They study genome wide association
• GWAS Searches the genome for genetic variants (SNPs)
• GWAS analyze thousands of nucleotide variants simultaneously

Array Comparative Genome Hybridization (aCGH)

• A process where patient and control DNA is labeled with fluorescent dyes
• The DNA is applied to a microarray
• It can scan a genome for imbalances
• It simultaneous detects aneuploidies, depletions and duplications

Analyzing Proteins

• Western blot (immunoblotting) detects polypeptides after size-fractionation of a tissue lysate on a polyacrylamide gel
• Immunohistochemistry (IHC) examines protein expression across tissue slices
• Immunoassays use antibodies to quantify protein or antigen amounts in lysates
• Assays include Radiolabelled (Radioimmunoassay (RIA)) and Enzyme linked (Enzyme linked immunosorbant assay (ELISA))

Dystrophin Protein Analysis in MD

• Immunohistochemistry assesses protein expression patterns
• It reveals the amount and cellular location of protein
• It doesn't not reveal the protein size
• Western blotting assesses the amount and size of protein instead
• Western blotting does not assess protein location

Western Blot

• This method helps determine the amount and size of proteins
• It can use samples from normal, Becker and Duchenne genetics
• Complete absence of the dystrophin protein indicates that the test subject had the severe disease, Duchenne