RECOMBINANT DNA TECHNOLOGY for

undergraduatesPart II

(PCR, DNA Fingerprinting, Blotting techniques)

Dr. Gangadhar ChatterjeeMBBS;MD

Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India

POLYMERASE CHAIN REACTION

EACTION

History OF PCR• As is the photo copier a basic

requirement in an office, so is the PCR machine in a molecular biology Laboratory !!!!!!!!!

• PCR is DNA replication in a test tube……..

• Great mind behind this PCR :Kary Banks Mullis

• Developed PCR in 1985 and was awarded Nobel prize in 1993.

• PCR machine otherwise called Thermocycler.

• 1983—Kary Mullis, a scientist working for the Cetus Corporation was driving along US Route 101 in northern California when he came up with the idea for the polymerase chain reaction.

• 1985—the polymerase chain reaction was introduced to the scientific community at a conference in October. Cetus rewarded Kary Mullis with a $10,000 bonus for his invention

• Later, during a corporate reorganization, Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-La Roche for $300 million.

PCR targets and amplifies a specific region of a DNA strand.

It is an invitro technique to generate large quantities of a specified DNA.

Often, only a small amount of DNA is available eg. A drop of blood, Semen strains, Single hair, vaginal swabs etc.

Two methods currently exist for amplifying the DNA or making copies

Cloning—takes a long time for enough clones to reach maturity

PCR—works on even a single molecule quickly

Requirements of PCR

DNA Template Primers Taq polymerase Deoxynucleoside triphosphates(dNTPs) Buffer solution Divalent

cations(eg.Mg2+ )

STEPS INVOLVED

DENATURATION:

The reaction mixture is heated to a temperature between 90-98° C so that the ds DNA is denatured into single strands by disrupting the hydrogen bonds between complementary bases.

Duration of this step is 1-2 mins.

ANNEALING

Temperature of reaction mixture is cooled to 45-60° C

Primers base pair with the complementary sequence in the DNA.

Hydrogen bonds reform. Annealing fancy word for renaturing.

EXTENSION:The temperature is now shifted to 72° C which is ideal

for polymerase.Primers are extended by joining the bases

complementary to DNA strands.

Elongation step continues where the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template.

Now first cycle is over and next cycle is continued ,as PCR machine is automated thermocycler the same cycle is repeated upto 30-40 times.

NEW AUTOMATED PCR

OLD PCR

Factors for Optimal PCR:PCR Primers-correctly designed pair of primers is required-primer dimer, hairpin formation should be prevented-length of primer

DNA Polymerase-Thermus aquaticus-170° F-Taq polymerase is heat resistant-It lacks proof reading exonuclease activity-Other polymerases can be used .eg:Tma DNA Polymerase from Thermotoga maritama, Pfu DNA Polymerase from Pyrococcus furiosus.

Annealing Temperature- Very important since the success and specificity of PCR

depend on it because DNA-DNA hybridization is a temperature dependent process.

- If annealing temperature is too high, pairing between primer and template DNA will not take place then PCR will fail.

- Ideal Annealing temperature must be low enough to enable hybridization between primer and template but high enough to prevent amplification of nontarget sites.

- Should be usually 1-2° C or 5° C lower than melting temperature of the template-primer duplex

Melting Temperature- Temperature at which 2 strands of the duplex

dissociate.It can be determined experimentally or calculated

from formulaTm = [4(G+C) + (2(A+T)]

G/C content- ideally a primer should have a near random mix

of nucleotides, a 50% GC content- there should be no PolyG or PolyC stretches

that can promote non-specific annealing

Variations of PCR:PCR is highly versatile technique and has been modified in variety of way to suit specific applications.Inverse PCR Anchored PCR Reverse transcriptase PCR

RACE PCR Quantitative real time PCR (Q-RT PCR) Real time PCR using DNA dyes Fluorescent reporter probe method Asymmetric PCR Allele- Specific PCR

Overlap extension PCR Assemble PCRHelicase dependent amplicationIntersequence-specific PCR(ISSR) Ligation-mediated PCRMethylation –specific PCRMiniprimer PCRMultiplex PCRNested PCRSolid phase PCRTouch down PCR

Limitations

Sequence InformationAmplicon size Error rate during amplificationSensitivity to inhibitorsContaminationArtifacts

DNA FINGERPRINTING

Each DNA profile is Unique

Alec Jeffreys, 1985

• On DNA sequence of nucleotide Is repeated again and again but differ for person to person

• When the number is not known, variable, or irrelevant, it is sometimes called a variable number tandem repeats (VNTR)

When between 10 and 60 nucleotides are repeated – Mini- satellite

Those with fewer are known as microsatellites or short tandem repeat

• Introns contain blocks of repeated nucleotides called short tandem repeats (STRs)

• It is the number of times that these STRs are repeated that produces the variations in individuals.

• DNA fingerprinting Is also called DNA profiling

• Up to 1984, the only method of establishing and authenticating personal identification was by the fingerprint process.

• DNA fingerprinting technique was devised in 1985 by Alec Jeffrey at University of Leicester in England, while working on the sequences within myoglobin gene.

Stages of DNA Fingerprinting

• DNA is extracted from sample• To extract DNA , most commonly used

detergent is Sodium Dodecyl Sulfate (SDS).• DNA in the nucleus of the cell is molded,

folded, and protected by Proteins

• Proteinase K is used to break up the proteins

Stages of DNA Fingerprinting

• Ethanol Precipitation

• The DNA must be purified from the cell extract.

Stages of DNA Fingerprinting

DNA cuts with the help of Restriction Endonuclease• DNA is cut into millions of small fragments with the help of

restriction endonucleases

• Restriction enzymes chop DNA at specific sequences.

• the patterns occur in different places in different individual , the length of the fragment differs from person to person.

e.g The EcoRI restriction enzyme recognizes the following sequence

-C-A-A-T-T-G- -G-T-T-A-A-C

Separation of DNA fragments

• DNA fragments are separated by electrophoresis

• DNA is negatively charged so it is attracted to the positive end of the gel.

Separated DNA on the basis of gel electrophoresis

Stages of DNA Fingerprinting

Southern blotting Patterns of fragments are transferred to a nylon membrane by a process called Southern blotting.

Stages of DNA Fingerprinting

DNA fingerprints compared • nylon or nitrocellulose

(From southern blot) sheet is placed under X-ray film

• The radioactive probes on the DNA fragments expose the film

• produces visible pattern of light and dark bands which is unique to each individual

Light and dark bands of DNA when nylon film exposed to X- ray

 

Methods of DNA FingerprintingRestriction enzyme based fingerprinting

• Relatively straight forward

• When genomic DNA is cleaved with particular restriction enzymes, the resulting fragments can contain a minisatellite (VNTR) region

• gel electrophoresis separates the digested DNA into bands based on the length of the fragments, the pattern of bands reflects the number of repeats in a minisatellite

• each person’s DNA will yield a unique banding pattern

Methods of DNA FingerprintingPCR – based DNA fingerprinting

•By virtue of the amplification process, it is possible to begin with only a tiny amount of DNA

• possible to fingerprint the DNA of a single cell

• Also possible to analyze old or degraded samples using PCR-based fingerprinting

• Degraded DNA - be nearly impossible to fingerprint with restriction analysis

Methods of DNA Fingerprinting

Amplified fragment Length PolymorphismCome in 90’s

remains attractive because of its relatively less complicated operation and the cost-effectiveness

use of gel in its analysis phase, there are issues of bunching of the VNTR's, causing misidentifications in the process.

Methods of DNA Fingerprinting

STR ( Short tandem repeats) Mostly widely used method for comparing samples of

persons

STR analyzes how many times base pairs repeat themselves on a particular location on a strand of DNA

Has been extremely successful for use in the personal identification of criminal suspects, paternity issues, as well as in identification of the diseases.

Applications of DNA fingerprinting

Mostly used to solve crime and medical, paternity problems

Violent murder occurred Rape cases Inheritance casesImmigration cases

DNA fingerprinting also found recent applications in food industry

Recent application in breast cancer detection

Applications of DNA Fingerprinting • Pattern of the DNA profile

is compared with those of the victim and the suspect

• profile matches the suspect - provides strong evidence that the suspect was present at crime scene

DIFFERENT BLOTTING TECHNIQUES

What is BLOTTING? Blotting is a technique for transferring DNA, RNA and

Proteins onto a carrier so they can be separated, and often

follows the use of a gel electrophoresis.

TYPES OF BLOTTING TECHNIQUES

Blotting technique

Southern Blot

It is used to detect DNA.

Northern Blot

It is used to detect RNA.

Western blot

It is used to detect protein.

SOUTHERN BLOTTING Professor Sir Edwin Southern,

Professor of Biochemistry and Fellow of Trinity, developed this method in 1975.

Southern won the Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences.

He developed this procedure at Edinburgh University more than 30 years ago. The technique is known as DNA transfer or 'Southern blotting‘. Professor Sir Edwin Southern

This method Involves Separation, Transfer and

Hybridization.

It is a method routinely used in molecular biology for

detection of a specific DNA sequence in DNA samples.

The DNA detected can be a single gene, or it can be part

of a larger piece of DNA such as a viral genome.

Southern blotting combines Agarose Gel

Electrophoresis for size separation of DNA with

methods to transfer the size separated DNA to a filter

membrane for probe hybridization.

The key to this method is Hybridization.

Hybridization - Process of forming a double-stranded

DNA molecule between a single-stranded DNA probe

and a single-stranded target patient DNA.

PRINCIPLE1. The mixture of molecules is separated.

2. The molecules are immobilized on a matrix.

3. The probe [a hybridization probe is a fragment of DNA or RNA of variable

length (usually 100-1000 bases long) which is radioactively labeled. It can then

be used in DNA or RNA samples to detect the presence of nucleotide sequences

(the DNA target) that are complementary to the sequence in the probe] is added to

the matrix to bind to the molecules.

4. Any unbound probes are then removed.

5. The place where the probe is connected corresponds to the location of the

immobilized target molecule.

CAPILLARY BLOTTING APPARATUS

Steps in southern blotting

1. Digest the DNA with an

appropriate restriction

enzyme.

2.The complex mixture of

fragments is subjected to gel

electrophoresis to separate the

fragments according to size.

3. The restriction fragments present in

the gel are denatured with alkali.

4. It is then transferred onto a

nitrocellulose filter or nylon

membrane by blotting.

This procedure preserves the

distribution of the fragments in the

gel, creating a replica of the gel on

the filter.

5.The filter is incubated under

hybridization conditions

with a specific radiolabeled

DNA probe.

The probe hybridizes to the

complementary DNA

restriction fragment. 

6.Excess probe is washed

away and the probe bound

to the filter is detected by

autoradiography, which

reveals the DNA fragment

to which the probe

hybridized.

APPLICATIONS Southern blots are used in gene discovery , mapping, evolution and

development studies, diagnostics and forensics (It is used for DNA

fingerprinting, preparation of RFLP [Restriction Fragment Length

Polymorphism] maps).

Identification of the transferred genes in transgenic individuals, etc. It allow

investigators to determine the molecular weight of a restriction fragment and

to measure relative amounts in different samples.

It is used to detect the presence of a particular bit of DNA in a sample.

Used to analyze the genetic patterns which appear in a person's DNA.

Northern Blotting

Northern blotting is a technique for detection of specific RNA

sequences. Northern blotting was developed by James Alwine

and George Stark at Stanford University (1979) and was

named such by analogy to Southern blotting.

Applications

A standard for the study of gene expression at the level of mRNA

(messenger RNA transcripts)

Detection of mRNA transcript size

Study RNA degradation

Study RNA splicing

Study RNA half-life

Often used to confirm and check transgenic / knockout mice (animals)

Western blotting

Western blotting (1981) is an Immunoblotting technique which

rely on the specificity of binding between a protein of interest

and a probe (antibody raised against that particular protein) to

allow detection of the protein of interest in a mixture of many

other similar molecules.

The SDS PAGE technique is a prerequisite for Western blotting .

Applications1.The confirmatory HIV test

2.Western blot is also used as the definitive test for

Bovine spongiform encephalopathy (BSE(

3.Some forms of Lyme disease testing employ Western

blotting.

“To be or not to be, That is the question.”

- The tragedie of Hamlet, Prince of DenmarkeWilliam Shakespeare

1602