molecular techniques

Plasmids, restriction enzymes, analytics

Plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.

Vector – a carrier (plasmid or other type) used for bringing target DNA fragment into a host cell.

Vector types

Vector Target fragment length

Plasmid 0-10 kb (total size up to 15 kb)

Cosmid 10-40 kb

P1 artificial chromosome (PAC) 130-150 kb

Bacterial artificial chromosome (BAC) About 300 kb

Yeast artificial chromosome (YAC) 200 kb to 2 Mb

Plasmids are essential instruments of molecular biology

Cloning and sequencing of DNA and cDNA fragments Generation of genomic and cDNA libraries Expression of recombinant proteins Generation of mutant proteins Analysis of regulatory sequences Gene targeting

Essential vector elements

Origin of replication Antibiotic resistance gene (Amp, Kan, Tet, Chl) (Multiple cloning site)

Map of pOTB7 vector showing Chloramphenicol resistance gene (CMR), replication origin (ORI) and multiple cloning site (MCS)

Optional plasmids elements

Multiple cloning site Promoter for cloned sequence Reporter gene Tag Regulatory sequences

pc DNA 3.1 ( +) EGF P6131 bp

Am p r

Neo

BHG polyASV40 poly A

P CM V

SV40 prom

T7

Apa I (170 5)

Bam H I (930)

Bgl II (13)

Bst X I (1678)

Eco R I (1656)

Eco R V (1668)

Ehe I (2969 )

H in dIII (912)

Kpn I (9 22)

Mlu I (229)

Nde I (4 85)

Not I (16 83)

Sca I (5689)

Sma I (2781)

Spe I (250)

X ba I (16 95)

X ho I (16 89 )

Important plasmid information

Replication origin defines the host bacteria: ColE1 replication origin is required for E.coli

Replication origin may define the number of plasmid copies per bacterial cell

Bacteria may lose recombinant plasmid during cultivation due to the absence of partitioning system (par). Naturally occurring plasmids contain par that ensures that every bacterial cell contains the plasmid.

Selection of the plasmid vectorCopy number

Replication origin Intended use

Replication origin of pBR322 vector restricts number of plasmid copies per cell to 30-40.

Expression of proteins in bacteria. Very useful for toxic protein or when tight control of protein amount per bacterial cell is needed.

Replication origin of pUC vector is a mutated version of pBR322 lacking Rop/Rom gene and allows up to 500 copies of plasmid per cell.

Amplification of high amounts of plasmid DNA in bacteria.Expression of high amounts of proteins in bacteria.

Selection of the plasmid vectorPurpose of use

Purpose Special vector feature(s) Example

Recombinant protein expression in bacteria

Regulated bacterial promoterTag for protein purification

pGEX4T

Recombinant protein expression in eukaryotic cells

Eukaryotic promoterTag for protein purification or detectionEukaryotic selection marker

pcDNA3.1

Analysis of eukaroytic promoter Reporter gene pGL3basic

General cloning - pBluescript KS

Restriction enzymes (endonucleases)

Cut specific DNA sequence Protect bacteria from phage infection by digesting phage

DNA after injection Cellular DNA is protected by methylation that blocks

restriction enzyme activity Restriction enzyme (RE) means restricts virus replication Endonucleases are enzymes that produce internal cut called

as cleavage in DNA molecule


Presence of RE was postulated in 1960 by W.Arber The first true RE was isolated in 1970 by Smith, Nathans and

Arber. In 1978 they were awarded the Nobel Prize for Phylsiology and Medicine.

RE remain indispensible from molecular cloning and sequencing.

Type I enzymes cut at a site that differs, and is located at least at at least 1000 bp away, from their recognition site.

Type II enzymes recognize sites of 4-8 nucleotides and cleave DNA at the same site

Type III enzymes recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.



Creating genomic and cDNA libraries Cloning DNA molecules Studying nucleotide sequence Generating mutated proteins

Plasmids, restriction enzymes, analytics

Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix.

Ethidium bromide stained agarose gel of total RNA (1-3) and DNA ladder (M)

Plasmid preparation stage 1

1. Plasmid-containing bacteria are cultivated in liquid media, supplemented with the antibiotics for 18 h at 37°C with intensive shaking

2. Cells are harvested by centrifugation

Preparation of the lysate

3 solutions strategy1. Resuspend in hypotonic buffer with RNase (buffer P1)2. Lyse bacteria using NaOH/SDS solution (buffer P2)3. Neutralize NaOH and precipitate proteins using NaAc buffer

(buffer P3)

Plasmid can be isolated from obtained lysate using various strategies.

Possible methods for isolation

1. Ethanol or Isopropanol precipitation2. Silica matrix bind-wash-elute procedure3. Density gradient centrifugation

Precipitation “quick and dirty”

1. Ethanol is added to the lysate2. Obtained sample incubated for 30 min3. DNA is collected by centrifugation

• Cheap• Fast

Advantages Disadvantages• Small amounts of DNA• Poor purity, not sufficient for

applications like transfection and in vitro translation

• Concentration of the plasmid can not be determined photometrically

Also known as mini prep

Silica matrix columns

1. Apply lysate on the column2. Wash the column3. Elute the plasmid4. Precipitate

• High purity of the plasmid• Fast

Advantages Disadvantages• Expensive

Gradient centrifugation

1. Mix lysate with CsCl solution2. Add EtBr3. Centrifuge in the ultracentrifuge for 12-36h4. Collect the plasmid5. Precipitate

• The very best plasmid purity• Relatively cheap

Advantages Disadvantages• Slow• Expensive equipment is needed• High concentrations of EtBr

Concentration measurement

Photometric measurement of DNA concentrationUV 260 nmConc=50xOD260

Important! Photometric measurement of DNA concentration can not be applied for “quick and dirty” plasmids, because of the presence of RNA rests.

Gel electrophoresis of plasmid DNA

Selection of agarose concentration

Plasmid on an agarose gel

Characteristics of Nucleic Acids

• Two types of nucleic acids: RNA & DNA• DNA is encoded with four interchangeable

"building blocks", called "bases", Adenine, Thymine, Cytosine, and Guanine, with Uracil rarely replacing Thymine

• RNA has five different bases: adenine, guanine, cytosine, uracil, and more rarely thymine.

Deoxyribonucleic Acid

DNA Replication• Replication is performed by splitting (unzipping) the double

strand down the middle via relatively trivial chemical reactions, and recreating the "other half" of each new single strand by drowning each half in a "soup" made of the four bases.

• Each of the "bases" can only combine with one other base, the base on the old strand dictates which base will be on the new strand.

• This way, each split half of the strand plus the bases it collects from the soup will ideally end up as a complete replica of the original, unless a mutation occurs.

DNA Replication

Nucleic Acid Probes• Spontaneous pairing of complementary DNA strands forms basis for

techniques used to detect and characterize genes.• Probe technology used to identify individual genes or DNA sequences.• Nucleic acid probe short strand of DNA or RNA of known sequence used

to identify presence of complementary single strand of DNA in patient sample.

• Binding of 2 strands (probe and patient) known as hybridization.• 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.

Dot-Blot

• Dot-blot clinical sample applied to membrane, heated to denature DNA.

• Labeled probes added, • Wash to remove unhybridized probe and

measure reactants.• Qualitative test only.• May be difficult to interpret.

Dot-Blot Hybridization

• Figure 1 DNA–DNA dot-blot hybridization between maize genomic DNA and a CaMV p-35S probe. Sample numbers coincide with those in ref. 1. Top row: 1, 100% transgenic; 2, 10% transgenic; 3, 5% transgenic; 4, 1% transgenic, 5, 0.5% transgenic; 6, historical maize negative control; 7, water negative control; 8, Diconsa sample K1. Bottom row: 1, criollo sample B1; 2, criollo sample B2; 3, criollo sample B3; 4, criollo sample A1; 5, criollo sample A2; 6, criollo sample A3; 7, Peru maize negative control P1; 8, water negative control.

Sandwich Hybridization

• Uses two probes, one bound to membrane and serves as capture target for sample DNA.

• 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• Restriction endonucleases cleave both strands of

double stranded DNA at specific sites, approximately 4 to 6 base pairs long.

• Further separated on the basis of size and charge by gel electrophoresis.

• Digested cellular DNA from patient/tissue added to wells in agarose gel and electric field applied, molecules move.

• Gel stained with ethidium bromid and vieuwed under UV light.

Sandwich Hybridization• Differences in restriction patterns referred to as restriction

fragment length polymorphisms (RFLPs)• Caused by variations in nucleotides within genes that change

where the restriction enzymes cleave the DNA.• When such a mutation occurs different size pieces of DNA are

obtained.• Caused by variations in nucleotides within genes that change

where the restriction enzymes cleave the DNA.• When such a mutation occurs different size pieces of DNA are

obtained.

Southern Blot• DNA fragments separated by electrophoresis.• Pieces denatured and transferred to membrane for

hybridization reaction.• Place membrane on top of gel and allow buffer plus DNA to

wick up into it.• Once DNA is on membrane heat or use UV ligth to crosslink

strands onto membrane to immobilize.• Add labeled probes for hybridization to take place.• Probes added in excess so target molecules reanneal and

more likely to attach to probe.

Southern Blot

• The Southern Blot takes advantage of the fact that DNA fragments will stick to a nylon or nitrocellulose membrane. The membrane is 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• 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 allow investigators to determine the molecular weight of an mRNA and to

measure relative amounts of the mRNA present in different samples. • RNA (either total RNA or just mRNA) is separated by gel electrophoresis, usually an agarose

gel. Because there are so many different RNA molecules on the gel, it usually appears as a smear rather than discrete bands.

• The RNA is transferred to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The RNA molecules retain the same pattern of separation they had on the gel.

• The 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).

• The location of the probe is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen or gives off light which will expose 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 tose using

chemiluminescent labels.• Assays performed in a few hours.

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.

Fluorescent In-Situ Hibridization FISH

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

information from humans and bacteria. • This utilizes the complementation of the four bases labeled A (adenine), T

(thymine), G (guanine) and C (cytosine) in which A pairs with T and G pairs with C through hydrogen bonding.

• A solution of DNA sequences containing known genes called a DNA probe is placed on glass plates in microspots several microm 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.

How DNA Chips Are Made

• Used to examine DNA, RNA and other substances• Allow thousands of biological reactions to be

performed at once.

Step 1: Make gene probes.• Using conventional techniques such as polymerase chain

reaction and biochemical synthesis, strands of identified DNA are made and purified. A variety of probes are available from commercial sources, many of which also offer custom production services.

Step 2: Manufacture substrate wafer.

• Companies use photolithography and other nanomanufacturing techniques to turn glass and plastic wafers into receptacles for the DNA probes.

Step 3: Deposit genetic sequences.

• Manufacturers use a variety of processes ranging from electrophoretic bonding to robotic deposition to adhere genetic material to the substrate. Cleanroom conditions and standards must be observed to attain the degree of contamination control needed during the deposition process.

DNA Chip

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

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, transcription mediated amplification,, strand displacement amplification and nucleic acid sequence-based amplification.

Polymerase Chain Reaction• Capable of amplifying tiny quantities of nucleic acid.• Cells separated and lysed.• Double stranded DNA separated into single strands.• 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.– 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 a 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.

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

Transcription Mediated Amplification

• TMA is the next generation of nucleic acid amplification technology.• 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.

QB Replicase• Uses an RNA directed RNA polymerase that replicates the

genomic RNA of a bacteriophage named QB.• The RNA genome of QB is essentially the only substrate

recognized by the polymerase.• Because a short probe can be inserted into the QB RNA this

becomes the system for amplification.• After the probe has annealed to the target, unbound probe is

treated with RNase and washed away.• The hybridized probe is RNase resistant.• When QB replicase is added the probe is enzymatically

replicated to detectable levels.

Ligase Chain Reaction• The LCR test employs four synthetic oligonucleotide probes to anneal at specific

target sites on the cryptic plasmid. • Each pair of probes hybridize close together on the target DNA template.• Once the probes are annealed, the gap is filled by DNA polymerase and close by

the ligase enzyme.• This two-step process of closing the gap between annealed probes makes the LCR,

in theory, more specific than PCR technology. • The ligated probe pairs anneal to each other and, upon denaturation, form the

template for successive reaction cycles, thus producing a logarithmic amplification of the target sequence.

• Like PCR, LCR is made in a thermocycler. • The LCR product is detected in an automated instrument that uses an

immunocolorimetric bead capture system. • At the end of the LCR assay, amplified products are inactivated by the automatic

addition of a chelated metal complex and a oxidizing agent.

Drawbacks of Amplification Systems

• Potential for false-positive results due to contaminating nucleic acids.

• PCR and LCR, DNA products main source of contamination.• QB replicase and 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

Signal Amplification• Replicates signal rather than either the target or the probe.• Based on the reporter group (the labeled tag) being attached in greater numbers to the probe

molecule or increasing the intensity generated by each labeled tag.• Patient nucleic acid not replicated or amplified technique is less prone to contamination.• Sensitivity is lower.• Branched chain signal amplification employs several simultaneous hybridization steps.• Author states similar to decorating a Christmas tree and involves several sandwich

hybridizations.– first, target specific oligonucleotide probes captures target sequence to solid support.– Second set of target specific probes called extenders hybridize to adjoining sequences and act as

binding site for large piece called branched amplification multimer.– Each branch has multiple side branches capable of binding numerous oligonucleotides.

• Branched chains are well suited to detection of nucleic acid targets with sequence heterogeneity such as hepatitis C and HIV.

molecular techniques

Science