Unit 7 - Techniques and Diagnostics Flashcards Preview

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DNA diagnostic tests

PCR

Reverse Transcriptase PCR (RT-PCR)

real time PCR
DNA sequencing and Next Generation DNA sequencing

DNA microarrays

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what is PCR named after

Taq DNA Polymerase from thermus aquaticus that is used to amplify a piece of DNA by in vitro enzymatic replication

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MOA of PCR

as PCR progresses, the DNA generated is itself used as a template for replication

This sets in motion a chain reaction in which the DNA template is exponentially amplified

With PCR it is possible to amplify, very specifically, a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece

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what is needed for PCR

DNA template that contains the DNA region (target) to be amplified

Two primers, forward and reverse, which are complementary to the DNA regions at the 5' or 3' ends of the DNA region

Taq polymerase with a temperature optimum at around 70°C

Deoxynucleotide triphosphates (dNTPs) the building blocks from which the DNA polymerases synthesizes a new DNA strand

Buffer solution which contains Mg2+ , providing a suitable chemical environment for optimum activity and stability of the DNA polymerase

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thermal cycling

alternately heating and cooling the PCR sample to a defined series of temperature steps

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3 steps of PCR procedure

  1. denaturation of the template DNA at 94°C
  2. Annealing of the single stranded primers at 55-65°C
  3. extension of the annealed primers by addition of nucleotides by base pairing to the template DNA at 72°C

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1st cycle

after 1 cycle of synthesis the rxn mixture is again heated to dissociate the DNA strands and cooled to re-anneal the DNA and primers

primers are extended again

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2nd cycle

2 newly synthesised, single stranded chains are precisely the length of the DNA between the 5' ends of the primers

 

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3rd cycle

2 double-stranded DNA molecules that exactly match the target sequence are produced

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what changes with each cycle

the number of DNA strands, whose 5' to 3' ends are defined by the ends of the primers, increases exponentially

As a result, the desired DNA is preferentially replicated until after 20-30 cycles, it makes up most of the DNA in the tube

(PCR products are visualized by agarose gel electrophoresis)

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what is Agarose Gel Electrophoresis

how does it work

agarose is a polysaccharide which acts as a molecular sieve

An electric current is applied across the gel

DNA is negatively charged and is attracted to the positive anode

The DNA is separated based on size – shorter molecules move faster through gel than longer

Gel contains ethidium bromide dye (or SYBR Green) to allow DNA to be seen under ultraviolet light

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advantages of PCR

time taken to amplify sufficient amounts of the target sequence

a single molecule of the target sequence can be amplified to 109 copies in 1.5-6 hours - In contrast, it takes several days to weeks to produce similar levels using cell-based approaches (i.e. plasmid vectors and host bacterial cells)

Sensitivity

A single copy of the target DNA sequence can be amplified rapidly to usable concentrations (e.g. can be visualized on a gel), hence the usefulness of PCR in forensic science

Robustness

PCR can be used to amplify target gene sequence information from partially degraded DNA samples or from tissues that have been formalin-fixed on slides

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disadvantages of PCR

1. Prior sequence knowledge is essential

forward and reverse primers are designed from known DNA sequence data

2. Limited size range of PCR products

PCR products are generally 200-100 bases in length (most accurate/reproducible range), although products of up to 5 kb have been amplified (rare)

3. DNA Replication may be inaccurate

in a standard PCR reaction using an ordinary Taq Polymerase preparation, as much as 40% of the products will contain some error in the nucleotide sequence

4. Contamination/False Positives

Contamination from the operator or previous PCR reactions can lead to false positives

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5 medical applications of PCR

1. Genetic testing for e.g. carriers of cystic fibrosis etc

2. Pre-natal testing - disease mutation DNA samples can be obtained by amniocentesis or chorionic villus sampling

3. Pre-implantation genetic diagnosis where individual cells of a developing embryo are tested for mutations

4. Tissue typing for organ transplantation - proposal to replace the traditional antibody-based tests for blood type with PCR-based tests

5. Diagnosis of Infectious Disease e.g. Human Immunodeficiency Virus or Human Papilloma Virus or Hepatitis

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how is PCR used in cancer diagnostics

Many forms of cancer involve alterations to genes e.g. proto-oncogenes are mutated to oncogenes

by using PCR-based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient

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PCR and the bcr-abl oncogene

how is bcr-abl formed

The bcr-abl oncogene is the result of a translocation of DNA sequences from human chr9 to chr22 (Philadelphia Chromosome)

PCR can be used to detect the bcr-abl oncogene and determine which variant of the gene is present

→ Produces a new fusion protein from BCR and ABL genetic sequences

This translocation and the bcr-abl tyrosine kinase are present in 95% of chronic myelogenous leukemia (CML)

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applications of RT-PCR

Reverse transcription polymerase chain reaction is widely used in the diagnosis of genetic diseases

semi-quantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression

to determine risk of re-occurence of breast cancer in patients with stage 1 or 2 node-negative breast cancer

(limited - only a number of genes can be amplified)

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Oncotype-DX

RT-PCR-based assay performed on RNA extracted from paraffin-embedded tumour tissue

determines the level of expression in 21 genes, 16 of which are cancer-related genes and 5 are control reference genes

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MOA of real time/quantitative PCR

monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR

Fluorescent label e.g. SYBR Green is added to the DNA during amplification process, and detected by the Real Time PCR Machine

Real-time PCR can be used quantitatively (quantitative real-time PCR) and semiquantitatively (i.e., above/below a certain amount of DNA molecules) (semiquantitative real-time PCR)

Real time PCR – COVID-19 Testing

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uses for Chain Termination (Sanger) DNA Sequencing

genome projects - easily automated

(other methods include Chemical Degradation Method and Pyrosequencing)

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steps in the Chain Termination (Sanger) DNA Sequencing

 

First step – annealing of a short oligonucleotide primer to the same position on each DNA molecule

Acts a primer for the synthesis of new DNA strand complementary to the template

The strand synthesis reaction is catalyzed by the enzyme DNA Polymerase

Requires four deoxyribonucleotides triphosphates (dNTPS) – dATP, dTTP, dCTP and dGTP

Also, a small amount of terminating nucleotides, dideoxynucleotide triphosphates (ddNTPS) – ddATP, ddTTP, ddCTP and ddGTP are required to produce DNA fragments

Each dideoxynucleotide is labeled with a different fluorescent marker

Polymerase enzyme does not discriminate between deoxy- and dideoxynucleotides

Once incorporated a dideoxynucleotide blocks further strand elongation because it lacks the 3’-hydroxyl group needed to form the connection with the next nucleotide

Because normal deoxynucleotides are present in larger amounts than the dideoxynucleotides, the strand synthesis does not always terminate close to the primer

The result is a set of new molecules, all of different lengths ending in a dideoxynucleotide which indicates a nucleotide A,C,G, or T that is present at the equivalent position in the template

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main events of Chain Termination (Sanger) DNA Sequencing

Incorporation of ddATP results in chains that are terminated opposite Ts in the template – generated a family of ‘A’ terminated molecules

Incorporation of other ddNTPs generates ‘C’, ‘G’ and ‘T’ families

Each dideoxynucleotide is labeled with a different fluorophore

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what happens during electrophoresis in Sanger DNA Sequencing

how is the information interpreted

During electrophoresis, the labeled molecules move past a fluorescence detector, which identifies which dideoxynucleotide is present in each band

The information is passed on to an imaging system

The DNA sequence is represented by a series of peaks, one for each nucleotide position

The sequence can be printed out or entered directly into a storage device for future analysis

Automated sequencers with 96 capillaries in parallel – average of 750 bp per experiment – 864 kb can be generated per machine per day but requires 24 hour support, robotic devices to prepare sequencing reactions and load sequencers, to generate sequence of an entire genome in weeks

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Next Generation Sequencing Technologies (NGST) types

Roche 454 Sequencing

Applied Biosystems/ SOLiD

Illumina Genome Analyzer

Helicos

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MOA of Roche - 454 Sequencing

Step 1: Preparation of an adapter ligated single stranded DNA library i.e. DNA fragments of the entire genome are ligated to adapters to which PCR primers are designed

Step 2: Individual DNA fragments are attached to beads via the adapters

Step 3: The DNA fragment on each bead is amplified by emulsion PCR (EmPCR) i.e. the beads are suspended in an oil emulsion which includes PCR regents including primers designed from the adapter sequences

Step 4: After amplification by PCR, the individual beads are distributed into the wells of a PicoTiter Plate™

Step 5: The DNA fragments on each bead are sequenced by pyrosequencing using a 454 Sequencer

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Pyrosequencing (put in steps)

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how has NGST helped in clinical practice

helped the discovery of DNA sequence variants with clinical significance

use of DNA sequence of diagnostic markers is entering into clinical practice for the detection of DNA sequence variants or small insertions or deletions in genes

somatic changes in DNA from tumour tissue but not present in normal tissue the same person

Oncogenic DNA sequence variants are useful diagnostic biomarkers and have provided specific molecular targets for cancer therapies e.g. Pancreatic Cancer Model

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interaction between genes and their product

1000s of genes and their product i.e. RNA and proteins in a given organism function in a complicated and harmonious way