Unit 2 Basic Immunologic ReactionsPart 7 Molecular Techniques

Terry Kotrla, MS, MT(ASCP)BB

Molecular Diagnostic Assays Very powerful tools. Quick supplies information to assist in

diagnosis and monitoring of diseases. Bacteria Viruses Genetic diseases

This technology is impacting every area of the clinical laboratory.

Molecular Techniques Techniques include

Enzymatic cleavage of nucleic acids Gel electrophoresis Enzymatic amplification of target sequences Hybridization with nucleic acid probes

Advantages and disadvantages of each will be discussed.

Gene Genes are located on 23

pairs of chromosomes. DNA is packed into genes. Genes are units of heredity DNA organized into long

structures called chromosomes, genes are pieces of DNA which correspond to a single unit of inheritance.

Two types of nucleic acids: RNA & DNA DNA carries genetic information within

chromosomes of each cell. Main role is long-term storage of information. Blueprint to construct other cell components

RNA Transcribed from DNA Central role is protein synthesis

DNA and RNA DNA is encoded with four interchangeable

"building blocks", called "bases", Adenine and Thymine, which pair together. Cytosine and Guanine, which pair together.

RNA has five different bases: Adenine and Uracil, which pair together. Cytosine and Guanine, which pair together.

Three Main Differences DNA is double stranded, RNA is single

stranded. DNA contains deoxyribose, RNA contains

ribose. Complementary base to adenine is thymine in

DNA and uracil in RNA.

RNA versus DNA Structure

DNA Replication DNA very stable. Loses conformational structure under

extremities of heat, pH or presence of destablizing agents.

Semi-conservative process, one strand acts as template to create exact copy.

Bonds holding strands together are weak.

DNA Replication – In-Vivo Strand “unzips”, hydrogen bonds between base pairs are

broken. Sequence of bases on strand serve as template to which

complementary bases are added. When process is complete 2 identical DNA molecules are

formed.

DNA Replication – In-Vitro Two steps

Denaturation – can use heat or alkaline solutions to break bonds and separate the two strands, if heat is used term is called “melting”.

Annealing – strands cool and complementary strands spontaneously rejoin.

This process is exploited in the laboratory.

Types of RNA One strand of DNA serves as template for messenger

or “mRNA”. mRNA carries information from DNA to ribosome. Uracil transcribed where Thymine would have occurred.

Transfer RNA (tRNA) transports amino acids to make proteins.

Ribosomal RNA (rRNA) acts as site of protein synthesis directed by mRNA.

RNA less stable and degrades more rapidly than DNA.

Hybridization Techniques Spontaneous pairing of DNA strands forms

the basis for detection and characterization of genes.

Probe technology used to identify individual genes or DNA sequences.

Nucleic Acid Probes Nucleic acid probe is a short strand of DNA

or RNA of known sequence Used to identify presence of complementary

single strand of DNA in patient sample. Binding of the patient strand with the probe is

known as hybridzation.

Nucleic Acid Probes Two DNA strands must share at least 16 to 20

consecutive bases of perfect complementarity to form stable hybrid.

Match occurring as a result of chance less than 1 in a billion.

Probes labeled with marker: radioisotope, fluorochrome, enzyme or chemiluminescent substrate.

Hybridization can take place in solid support medium or liquid.

Solid Support Hybrization Dot-blot Sandwich hybridization Gel electrophoresis Southern Blot Northern Blot

Dot-Blot Dot-blot clinical sample applied to

membrane, heated to denature DNA. Labeled probes added and will bind to target

if present. Wash to remove unhybridized probe. Detect by autoradiography or enzyme assay. Qualitative test only. May be difficult to interpret.

Dot-Blot Hybridization

Sandwich Hybridization Uses two probes, one bound to membrane and serves

as capture target for sample DNA of interest. Second probe anneals to different site on target DNA

and has label for detection. Sample nucleic acid sandwiched between the two. Two hybridization events occur, increases

specificity. Can be adapted to microtiter plates.

Sandwich Hybridization

Characterization of DNA Restriction endonucleases cleave DNA at

SPECIFIC recognition sites approximately 4 to 6 base pairs long. Enzyme which cuts ds or ss DNA at specific site. Enzymes found naturally. Over 3000 identified, 500 available

commercially. Human DNA can yield millions of unique

fragments.

Characterization of DNA

EcoRI popular restriction endonuclease. Cuts wherever there is a G-C or C-G bond. Fragments separated based on size and electrical charge by

electrophoresis. Kits developed to detect specific gene fragments associated

with diseases or conditions

Characterization of DNA Differences in restriction patterns referred to

as restriction fragment length polymorphisms (RFLP).

Caused by variations in nucleotides within genes that change where restriction enzymes cleave DNA.

When mutations occur different sized pieces of DNA are obtained.

Use of RFLP Identify specific microorganisms Detect polymorphism in MHC genes. DNA fingerprinting – first method used Localization of genes for genetic disorders Determination of risk for disease Paternity testing

Use of RFLP

Southern Blot Used to detect a specific DNA sequence in a

sample. Use restriction endonuclease to cut DNA into

fragments. DNA fragments separated by electrophoresis.

Electrophoresis to Separate Fragments

Southern Blot Pieces denatured and transferred from gel to membrane for

hybridization reaction. Place membrane on top of gel, add weights or use suction and

allow buffer plus DNA to wick up onto the membrane. Once DNA is on membrane heat or use UV light to crosslink

strands to permanently attach the DNA onto membrane. Add labeled DNA probes for hybridization to take place. Radioactive, fluorescent or chromogenic dye used as label Probes added in excess so target molecules reanneal and

more likely to attach to probe. Visualize results based on label used.

Southern Blot

Southern Blot takes advantage of the fact that DNA fragments will stick to a nylon or nitrocellulose membrane.

Membrane laid on top of the agarose gel and absorbent material (e.g. paper towels or a sponge) is placed on top.

With time, the DNA fragments will travel from the gel to the membrane by capillary action as surrounding liquid is drawn up to the absorbent material on top.

After the transfer of DNA fragments has occurred, the membrane is washed, then the DNA fragments are permanently fixed to the membrane by heating or exposing it to UV light.

The membrane is now a mirror image of the agarose gel.

Southern Blot

Southern Blot http://www.koreanbio.org/Biocourse/index.php/Hybridization

Southern Blot

MOM [blue], DAD [yellow], and their four children: D1 (the biological daughter), D2 (step-daughter, child of Mom and her former husband [red]), S1 (biological son), and S2 (adopted son, not biologically related [his parents are light and dark green]).

Northern Blot Northern blots used to detect RNA in a sample. RNA (either total RNA or just mRNA) separated by gel electrophoresis. RNA transferred to sheet of nitrocellulose, though other membranes can

be used and immobilized by heat or UV light. Blot is incubated with a probe which is single-stranded DNA. This probe will form base pairs with its complementary RNA sequence

and bind to form a double-stranded RNA-DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it (e.g. alkaline phosphatase or horseradish peroxidase).

Location of the probe is revealed by incubating with a substrate that the attached enzyme converts to a colored product that can be seen or gives off light when exposed on X-ray film.

If the probe was labeled with radioactivity, it can expose X-ray film directly.

Northern Blot

Solution Hybridization Both target nucleic acid and probe free to interact in

solution. Hybridization of probe to target in solution is more

sensitive than hybridization on solid support Requires less sample and is more sensitive. Probe must be single-stranded and incapable of self-

annealing. Fairly adaptable to automation, especially those

using chemiluminescent labels. Assays performed in a few hours.

Solution Hybridization Second method is hybridization protection assay. Chemiluminescent acridinium ester attached to

probe as label. After hybridization solution subjected to alkaline

hydrolysis. Causes hydrolysis of ester if probe not attached to

target. If probe attaches to target light given off.

Solution Hybridization

In-Situ Hybridization Target nucleic acid found in intact cells. Provides information about presence of

specific DNA targets and distribution in tissues.

Probes must be small enough to reach nucleic acid.

Radioactive or fluorescent tags used. Requires experience.

In-Situ Hybridization Fluorescent in-situ hybridization (FISH) very

popular. Cytogenetic technique used to detect and

localize presence or absence of DNA sequences on chromosomes.

Used in genetic counseling, medicine and species identification.

FISH

FISH A metaphase cell positive for the bcr/abl rearrangement

(associated with chronic myelogenous leukemia) using FISH. The chromosomes can be seen in blue. The chromosome that

is labeled with green and red spots (upper left) is the one where the wrong rearrangement is present.

FISH DNA probes specific to regions of particular chromosomes are attached to

fluorescent markers and hybridized with a chromosome spread. Picture shows a computer-generated "false colour" image, in which small

variations in fluorescence wavelength among probes are enhanced as distinct primary colours.

The combination of probes that hybridize to a particular chromosome produces a unique pattern for each chromosome. This makes it particularly easy to detect segmental deletions and translocations among chromosomes.

DNA Chip aka Microarrays A DNA chip (DNA microarray) is a biosensor which analyzes gene

information from humans and bacteria. Utilizes the complementation of the four bases labeled A, T, G and C in

which A pairs with T and G pairs with C through hydrogen bonding. Solution of DNA sequences containing known genes (DNA probe) placed

on glass plates in microspots several microns in diameter arranged in multiple rows.

Genes are extracted from samples such as blood, amplified and then reflected in the DNA chip, enabling characteristics such as the presence and mutation of genes in the test subject to be determined.

As gene analysis advances, the field is gaining attention particularly in the clinical diagnosis of infectious disease, cancer and other maladies.

Microarrays Microarrays are a significant advance both because they may

contain a very large number of genes and because of their small size.

Useful in surveying large number of genes quickly or when the sample to be studied is small.

May be used to assay gene expression within a single sample or to compare gene expression in two different cell types or tissue samples, such as in healthy and diseased tissue.

Because a microarray can be used to examine the expression of hundreds or thousands of genes at once, it promises to revolutionize the way scientists examine gene expression.

This technology is still considered to be in its infancy.

Microarray Fix the single stranded DNA to chip. Labeled with different colors.

Microarray A microarray scanner provides a picture of what spots showed up green,

red, or yellow. A green dot would represent a gene that is expressed more in healthy cells

or have less expression in cancer cells, and a red dot represents a gene that is expressed more in cancer cells.

A yellow spot would be a gene that is expressed in both cancer and healthy cells. 

Microarray

Drawbacks Stringency, or correct pairing, is affected by:

Salt concentration Temperature Concentration of destabilizing agent such as formamide

or urea. If conditions not carefully controlled mismatches

can occur. Patient nucleic acid may be present in small

amounts, below threshold for probe detection. Sensitivity can be increased by amplification: target,

probe and signal

DNA Sequencing Method to determine the exact order of the

nucleotide bases in DNA. Unknown DNA sequences compared to

known. Several methods available. Sanger method of choice.

Sanger Method of DNA Sequencing Requires ss DNA template, DNA primer, DNA polymerase,

labeled nucleotides and modified nucleotides to terminate DNA elongation.

DNA sample divided into 4 separate reactions to normal (NTP) and ONE type of dideoxynucleotides (ddNTP) A, T, C or G are added.

ddNTPs will prevent addition of further nucleotides. Creates DNA strands of discrete sizes. Each reaction loaded on separate lane on gel and

electrophoresed. Sequence of nucleotides read in order to determine DNA

sequence.

DNA Sequencing

Sanger’s DNA Sequencing Excellent explanation with animations – MUST see.

http://www.dnalc.org/view/15922-Early-DNA-sequencing.html

Target Amplification In-vitro systems for enzymatic replication of target

molecule to detectable levels. Allows target to be identified and further

characterized. Examples:

Polymerase chain reaction (PCR) Transcription mediated amplification (TMA) Strand displacement amplification (SDA) Nucleic acid sequence-based amplification (NASBA)

Polymerase Chain Reaction Capable of amplifying tiny quantities of nucleic acid. Cells separated and lysed. Double stranded DNA separated into single strands (denatured). Primers, small segments of DNA no more than 20-30 nucleotides long, added. Primers are complementary to segments of opposite strands of that flank the target

sequence. Only the segments of target DNA between the primers will be replicated. Each cycle of PCR consists of three cycles:

Denaturation of target DNA to separate 2 strands by heating. Annealing step in which the reaction mix is cooled to allow primers to anneal to target

sequence Extension reaction in which primers initiate DNA synthesis using a DNA polymerase.

These three steps constitute ONE thermal cycle Each PCR cycle results in a doubling of target sequences and typically allowed to

run through 30 cycles, one cylce takes approximately 60-90 seconds.

Taq Taq polymerase ("Taq pol") is a thermostable

polymerase isolated from thermus aquaticus, a bacterium that lives in hot springs and hydrothermal vents.

"Taq polymerase" is an abbreviation of Thermus Aquaticus Polymerase.

Often used in polymerase chain reaction, since it is reasonably cheap and it can survive PCR conditions.

Polymerase Chain Reaction http://www.dnalc.org/resources/animations/pcr.html

Polymerase Chain Reaction

Polymerase Chain Reaction Sample with all test

components mixed together.

Put in thermocycler which cycles the temperature for each stage of the reaction.

Electrophorese to separate and identify.

Transcription Mediated Amplification Two Types to discuss:

Nucleic acid sequence-based amplification (NASBA)

Transcription mediated amplification (TMA)

Nucleic Acid Sequence-Based Amplification (NASBA) Alternative amplification procedure. One step isothermal process for amplifying

RNA. NASBA has proven to be successful in

detection of both viral and bacterial RNA in clinical samples.

NASBA The NASBA technique has been used to develop

rapid diagnostic tests for several pathogenic viruses with single-stranded RNA genomes: influenza A foot-and-mouth disease virus severe acute respiratory syndrome (SARS)-associated

coronavirus Human bocavirus (HBoV) Parasites like Trypanosoma brucei

Transcription Mediated Amplification Another alternate amplification method. TMA is an RNA transcription amplification system using two enzymes

to drive the reaction: RNA polymerase and reverse transcriptase. TMA is isothermal; the entire reaction is performed at the same

temperature in a water bath or heat block. This is in contrast to other amplification reactions such as PCR or LCR that require a thermal cycler instrument to rapidly change the temperature to drive the reaction.

TMA can amplify either DNA or RNA, and produces RNA amplicon, in contrast to most other nucleic acid amplification methods that only produce DNA.

TMA has very rapid kinetics resulting in a billion fold amplification within 15-30 minutes.

TMA Tests have been developed for:

Chlamydia trachomatis Neisseria gonorrhoeae Mycobacterium tuberculosis Mycobacterium avium HIV-1 HBV HCV

Strand Displacement Amplification An isothermal process that permits 1010-fold

amplification of a DNA target sequence in as little as 15 min.

Method for producing abundant DNA from valuable and often limited clinical specimens

Isothermal nature of the reaction process offers distinct advantages with regard to cost and simplicity of instrumentation.

Universal detection format permits the use of the same fluorescent detector probes across multiple analytes.

Strand Displacement Amplification Used for detection of:

Neisseria gonorrhoea Chlamydia trachomatis Mycoplasma pneumoniae Mycobacteria tuberculosis

Ligase Chain Reaction Method of DNA amplification. Amplifies the nucleic acid used as a probe. Two probes are used per each DNA strand and are

ligated together to form a single probe. Uses both a DNA polymerase enzyme and a DNA

ligase enzyme to drive the reaction. Requires thermocycler. Each cycle results in doubling of target. Greater specificity than PCR.

Ligase Chain Reaction Used for

Detection of single base mutations in genetic diseases.

Neisseria gonorrhoea Chlamydia trachomatis

Drawbacks of Amplification Systems Potential for false-positive results due to contaminating

nucleic acids. PCR and LCR, DNA products main source of contamination. TMA, RNA products are possible contaminants. Must have product inactivation as part of QC program. Separate preparation areas from amplification areas and use

of inactivation systems such as UV light help alleviate contamination.

Very expensive. Closed system, automation will also decrease number of

problems.

Future of Molecular Diagnostic Techniques

Despite expense may be times that rapid diagnosis will result in decreased cost.

Example: Mycobacteria - quick diagnosis no need for expensive respiratory isolation.

Detection of multi-drug resistant M. Tuberculosis will lead to more timely public health measures.

Incredibly useful in serology and microbiology. Increased specificity and sensitivity of molecular testing will become the

standard of practice in immunology and microbiology. Testing will continue to become more rapid as assays are automated

which will also bring down the costs. Author states will not replace culture for routine organisms, but it already

is, and as DNA chip technology improves, the ability to test for multiple organisms will become easier

References http://www.brc.dcs.gla.ac.uk/~drg/courses/bioinformatics_mscIT/slides/slides2/sld001.htm

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GelBlotting.html http://www.bioteach.ubc.ca/MolecularBiology/IdentifyingDNA/ http://www.bio.davidson.edu/courses/genomics/Front/surfingenomics.html http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm

http://www.cdc.gov/ncidod/eid/vol7no2/pfaller.htm