Session 9: Molecular Techniques and Diagnosis

Name some molecular techniques for analysis of DNA/RNA

PCR

DNA/RNA electrophoresis

Restriction analysis

DNA sequencing

Karyotyping

FISH

DNA hybridization

Name some molecular techniques for analysis of protein

Protein electrophoresis

Immunoassays

Enzyme assays

Mass spectrometry

Describe steps of PCR

1) Denaturation - use heat to separate the strands

2) Annealing - cooling and adding primers

3) Elongation - add heat-stable DNA polymerase

This process is repeated 20-30x until the specific sequence is present at 10^6 copies

Components of PCR reaction

DNA template

DNA primers

DNA polymerase enzymes (taq*

dNTPs

Buffer

Clinical application of PCR

1) Forensic medicine (from small sample of hair, single cell)

2) Detection of viral, bacterial and protozoal agents

3) Diagnosis and genetic analysis of inherited disease

4) Diagnosis and analysis of neoplastic disorders

5) Prenatal and pre-implantation diagnosis

Examples of clinical application of PCR: inherited diseases

- Beta-thalassemia

- Sickle cell disease

- Hemophilia

- Tay-Sachs disease

Examples of clinical application of PCR: neoplastic disorders

- Chronic myelogenous leukemia (CML)

- Acute lymphocytic leukemia (ALL)

- Follicular lymphomas

- Various other cancers

- Detecting activated oncogenes

Example of application of multiplex PCR

Cystic fibrosis (CF)

Multiplex PCR

Multiple primers for multiple targets - good for analysing diseases which are associated with multiple alleles e.g., CF

DNA/RNA gel electrophoresis is used to separate...

Charged molecules such as DNA, RNA and proteins according to their size

When electric current is passed through the DNA/RNA gel during electrophoresis - what happens?

Charged molecules move through the permeable gel matrix when electric current passes through it.

- Smaller molecules migrate through gel quickly - travel further

- Larger molecules migrate through gel slowly - travel shorter distance

Restriction analysis is based on ___ ___ enzymes produced by bacteria

restriction endonucleases (RE)

Restriction endonucleases recognise and cut specific DNA sequences called '___ ___' (in restriction analysis)

restriction site

Restriction analysis is used alongside DNA gel electrophoresis to...

- Investigate mutations

- Investigate DNA variation

- Clone DNA

Restriction Fragment Length Polymorphisms (RFLPs)

Differences in DNA sequence on homologous chromosomes that can result in different patterns of restriction fragment lengths (DNA segments resulting from treatment with restriction enzymes).

Two examples of diseases which have clinical use for Restriction Fragment Length Polymorphism (RFLP)

1) Sickle cell anemia = single base mutation removes restriction site within a gene

2) Junctional Epidermolysis Bullosa (JEB) = single base mutation creates restriction site within a gene

Sanger DNA sequencing

- Dideoxynucleotides (ddNTPs) halt DNA polymerization at each base

- Generates sequences of various lengths that encompass the entire original sequence.

- Terminated fragments are electrophoresed and the original sequence can be deduced

Benefits of Sanger DNA sequencing

- Cost-effective for sequencing single genes (99.99% accuracy)

- Works best for determining sequence of nucleotide bases in piece of DNA (less than 1,000 bp in length)

Components of a Sanger sequencing reaction

1) Thermostable DNA Polymerase

2) ddNTPs - dideoxynucleotides

3) dNTPs - nucleotides

4) Buffer

Three main steps for Sanger sequencing process

1) Chain-termination PCR with fluorescent, chain-terminating ddNTPs

2) Size separation by capillary gel electrophoresis

3) Laser excitation and detection by sequencing machine to produce output chromatogram

Human Genome Sequencing

The sequencing of the first human genome.

Composite "everyman" was assembled from the DNA of several volunteers

Becoming more common now - cheaper and more accessible

Next Generation Sequencing (NGS)

Any of the high throughput approaches to DNA sequencing using the concept of 'massively parallel processing'

Benefits of NGS vs Sanger

Advantages of NGS include...

- Higher sensitivity to detect low-frequency variants

- Faster turnaround time for high sample volume

- Comprehensive genomic coverage

- Ability to sequence hundreds-thousands of genes or gene regions simulataneously

1,000 Genomes Project

2008

- International effort to catalogue 'common' human genetic variation

- Sequence 1,000 genomes from people who declared themselves to be healthy

The goal of the 1,000 Genomes Project

Find common genetic variants with frequencies of at least 1% in populations studied

10,000 Genomes Project

2010

- International effort to catalogue 10,000 genomes (4000 healthy; 6000 diseased)

- Included rare inherited disorders

- Included more common disorders - obesity, autism, schizophrenia

- Showed contribution of rare variants to range of diseases

- Discovered new genetic variants and genes underpinning disease risk

100,000 Genomes Project

2012

- 100,000 genomes sequenced from 85,000 people

- Participants = NHS patients with rare diseases, their families and patients with cancer

Karyotyping

The process of pairing and ordering all the chromosomes of an organism

What is karyotyping used to detect?

- Changes in chromosome number

- Changes in chromosome structure e.g., chromosomal deletion, duplication, translocation, inversions

Karyotypes are prepared from mitotic cells that have been arrested in ___ or ___ of the cell cycle

metaphase or prometaphase

Why do karyotypes use cells that have been arrested specifically within metaphase or prometaphase?

Chromosome are in their most condensed form

Variety of tissue types can be used as a source of these mitotic chromosomes for karyotyping - name some

- Peripheral skin

- Skin biopsy

- Tumor biopsies

- Bone marrow biopsy (cancer)

For prenatal diagnosis, the source of mitotic cells (chromosomes) for karyotyping can be obtained by - what two procedures?

1) Amniotic fluid = amniocentesis

2) Chorionic villus biopsy

Two types of banding used for karyotyping which produce visible karyotype by staining the condensed chromosomes?

1) Giemsa banding = using giemsa stain

2) Quinacrine banding = fluorescent staining using quinacrine

Most common chromosome banding method for karyotyping?

Giemsa banding

What is the purpose of FISH (fluorescent in situ hybridization)?

FISH detects and localizes specific DNA sequences using fluorescently labeled complementary DNA probes

Steps of FISH

1) Target DNA = fluorescently labeled probe DNA

2) Denature = separate DNA strand (allow probe access)

3) Hybridize = bind probe to target

4) Analyze = analyze probe signals using fluorescent microscopy

Why is denaturation important step in FISH?

Allow the probe access to the target DNA to hybridize

What is the purpose of FISH? What does it investigate?

1) Investigate genes in situ e.g., deletion, duplication, amplification

2) Investigate chromosome structure e.g., deletions, duplications, translocations

3) Investigate chromosome number e.g., trisomy 21

DNA microarray

Research tool used to study gene expression

- Resulting grid of probes can hybridize to complementary 'target' sequences derived from samples to determine the expression level of specific m-RNAs in a sample

DNA microarray technology

Collection of synthetic DNA sequences (probes) attached to a spot, on a solid surface - research tool used to study gene expression

Applications of DNA microarray technology

- Investigate disease associated SNPs (SNP arrays)

- Investigate chromosome deletions/duplications (comparative genome hybridization assay)

- Investigate conditional gene expression e.g., compare gene expression in healthy tissue vs tumor

Name some molecular techniques used for the analysis of proteins

1) Protein electrophoresis

2) Immunoassays

3) Enzyme assays

4) Mass spectrometry

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

What does SDS-PAGE do?

Analytical technique used to separate proteins based on their molecular weight

Some applications of SDS-PAGE

1) Estimate relative molecular mass = determine relative abundance of major proteins in a sample

2) Assess purity of protein samples

3) Detecting rare proteins

4) Specialized staining methods (e.g., western blotting, 2D-electrophoresis and peptide mapping) can be used to detect scarce gene products and detect/separate isozymes of proteins

Specialized staining methods of SDS-PAGE such as western blotting, 2D-electrophoresis and peptide mapping are valuable as they can be used to...

1) Detect extremely scarce gene products

2) Detect and separate isozymes of proteins

Protein Gel Electrophoresis importance in sickle cell anaemia

Used to detect alloenzyme variation e.g., Sickle Cell Anemia

- Hemoglobin replacement of negatively-charged Glu in normal HbA beta-globin to a → neutral valine in HbS

- This change results in a protein with a slightly reduced negative charge

Antibody (immunoglobulin)

Y-shaped protein produced by B cells of the immune system in response to the presence of antigens on foreign body invaders e.g., viruses, bacteria, fungi, parasites

Antibodies are used in...

1) Diagnostic tests e.g., autoimmune disorders, infectious/metabolic diseases

2) Therapies e.g., recombinant antibody therapy for MS

ELISA

Enzyme-Linked Immunosorbent Assay

What is ELISA?

Plate-based assay technique used for detecting and quantifying soluble substances such as peptides, proteins, antibodies and hormones

Brief description of how ELISA is carried out

1) Antigen immobilized on solid surface (microplate)

2) Immobilized antigen is then complexed with an antibody that is linked to a reporter enzyme in DIRECT ELISA

3) Activity of reporter enzyme is measured via incubation with appropriate substrate to produce measurable product

Three types of ELISA

1) Direct

2) Indirect

3) Sandwich

Some examples of the clinical applications of ELISA

1) Identification of idiopathic thrombocytopenic purpura = detection of platelet antibodies in serum

2) Identification of systemic lupus (SLE) = detection of platelet antibodies in serum

3) HIV testing

4) Pregnancy testing (hCG in urine)

Enzyme Assays

Measure the activity of clinically useful enzymes

- Need high concentration substrate

- Amount of enzymatic reaction product is measured

In enzyme assays - the measurable product is the...

Amount of enzymatic reaction product

Example of enzyme assays in medicine

Liver Function Tests (LFT)

- Aspartate aminotransferase (AST)

- Alanine aminotransferase (ALT)

- AST/ALT ratio

- Alkaline phosphatase (ALP)

Other enzyme assays used in medicine

- Amylase/lipase = marker for pancreatitis

- G-Glutamyl transferase = markers for liver

- Acid phosphatase = marker for prostate cancer

Mass Spectrometry (MS)

Analytical technique used to measure mass-to-charge ratio (m/z) of one or more proteins in a sample

What does a mass spectrum plot show?

The mass-to-charge (m/z) ratio of ions present in a protein sample

Each peak in a mass spectrum shows

Component of unique mass/charge ratio in sample

Height of peaks in mass spectrum shows

Abundance of various components in sample

Mass spectrum plots are plotted against ___

intensities

Mass spectrophotogemtry can be used to identify...

1) Molecular weight

3) Quantity of known compounds

4) Determine structure and chemical properties of molecules

Sickle cell disease analytical technique

Sickle cell is looking at the analyte hemoglobins (Hb) which is a protein...

- Mass spectrometry

- Electrophoresis

- High-performance liquid chromatography (HLPC)

Cystic fibrosis (CF) disease analytical technique

Cystic fibrosis is looking at the analyte immunoreactive trypsinogen (IRT)...

- ELISA

Hypothyroidism disease analytical technique

Hypothyroidism is looking at the analyte thyroid-stimulating-hormone (TSH)...

- ELISA

Phenylketonuria (PKU) disease analytical technique

Phenylketonuria is looking at the analyte Phenylalanine (Tyrosine)...

- Mass spectrometry

- Enzyme assay

Maple syrup urine disease (MSUD) analytical technique

MSUD is looking at the analyte Leucine, Isoleucine, alloisoleucine

- Mass spectrometry

Isovaleric acidaemia (IVA) analytical technique

IVA is looking at the analyte Isovalerylcarnitine C5

- Mass spectrometry

Glutaric aciduria analytical technique

Glutaric aciduria is looking at the analyte glutarylcarnitine C5-DC

- Mass spectrometry

Homocystinuria analytical technique

Homocystinuria is looking at the analyte Methionine (homocysteine)...

- Mass spectrometry

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) analytical technique

MCADD is looking at the analyte Octanoylcarnitine C8 (decanoylcarnitine C10)...

- Mass spectrometry