Chapter 6 Molecular Biology Techniques

Acknowledgement
Centers for Disease Control and Prevention- Ethiopia (CDC-E)

American Society for Clinical Pathology (ASCP)

Addiss Ababa University University of Gondar University of Hawassa Jimma University Haromaya University

Learning objectives At the end of these chapter, the students will be able to: Discuss about Extraction and purification of nucleic acids. Discuss molecular techniques & their application Perform some of the basic molecular techniques & interpret

the results
Describe genome sequencing techniques

Outline

1. Isolation & extraction of DNA

2. Electrophoresis 3. PCR (polymerase chain reaction) 4. Hybridization tests
northern blotting southern blotting western blotting Dot and slot blotting FISH (fluorescent in situ hybridization) Microarrays

5. DNA sequencing
6. DNA finger printing(DNA typing or DNA profiling) 7. Review question 8. References

Isolation & extraction of DNA

DNA isolation & purification is a technique used in molecular biology laboratories. Many procedures like some types of PCR, require high quality, purified DNA. There are a number of standardized techniques and variations, which can be adopted according to the type of cells or tissues. Isolation and purification methods used in earlier times were lengthy and tiresome with the use of ultra-centrifugation. But now with the advancement of separation techniques, the procedure is very simple and short.

 In any method of extraction and purification, there are three

main steps: 1. Breaking of the cells 2. Removing debris 3. Extraction of DNA

4. Purification

Breaking of the cells :

One common method for lysis of bacterial cell is alkaline lysis buffer. In the case of animals, cells can be lysed by simple detergents or by hypotonic solutions.

Plant tissues can be homogenized by strong detergents such as SDS (sodium dodecyl sulphate) and heating at high temperatures. But there are various types of DNA isolation kits marketed by a number of biotechnology companies, which are very simple, short, and easy to handle.

Isolation of plasmid DNA

Genomic DNA Isolation from Blood: Genomic DNA isolation is performed according to the standard protocol suggested by Federal Bureau of Investigation, USA. Lysis of RBC After the blood samples (stored at -70C in EDTA vacutainer tubes) are thawed, a standard citrate buffer is added, mixed, and the tubes are centrifuged.

The top portion of the supernatant is discarded and additional buffer is added, mixed, and again the tube is centrifuged.
Lysis of WBC After the supernatant is discarded, the pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is incubated at 55C for one hour.

Purification The sample then is phenol-extracted once with a phenol/ chloroform/ isoamyl alcohol solution, and after centrifugation the aqueous layer is removed to a fresh microcentrifuge tube. Precipitation The DNA is ethanol-precipitated, resuspended in buffer, and then ethanol-precipitated a second time. Analysis of DNA After the pellet is dried, buffer is added and the DNA is

resuspended by incubation at 55C overnight, and the genomic

DNA solution is assayed by the polymerase chain reaction.

 The DNA, isolated and purified by any of these methods, can be used for a variety of experimental purposes: It can be used for restriction digestion analysis, cloning, ligation, transformation experiments, in vitro transcription, PCR amplification, RFLP (restriction length polymorphism), fingerprinting, sequencing, nick translation and radio labeling, preparation of genomic DNA library and cDNA library, etc.

Methods for Determining DNA Yield and Purity

DNA yield can be assessed using four different methods: o Absorbance (optical density) o Agarose gel electrophoresis o Fluorescent DNA-binding dyes o A luciferase-pyrophosphorylation-coupled quantification system

 The most common technique to determine DNA yield and purity is also the easiest methodabsorbance All that is needed for absorbance measurement is a spectrophotometer equipped with a UV lamp UV-transparent cuvettes (depending on the instrument) a solution of purified DNA. Absorbance readings are performed at 260nm (A260) where DNA absorbs light most strongly, and the number generated allows one to estimate the concentration of the solution. To ensure the numbers are useful, the A260 reading should be between 0.11.0.

 DNA itself, and most of the common contaminants found in DNA preps, have absorbances in the region 230nm to 320nm so measurement of the absorbances in this region allows measurement of the DNA concentration and provides information about the contaminant levels.
While we perform a measurements , the most important wave length to note are, 230nm: Guanidium salts (used to facilitate DNA binding to silica columns) and phenol (used in phenol/chloroform extractions) absorb strongly at 230nm, therefore high absorbances at this wavelength can be indicative of carryover of either of these compounds into the sample.

 260nm: DNA absorbs light most strongly at 260nm so the absorbance value at this wavelength (called A260) can be used to estimate the DNA concentration. 280nm: Since tyrosine and tryptophan residues absorb strongly at this wavelength, the absorbance at 280nm is used as an indicator of protein contamination. 320nm: A320 provides a general measurement of the turbidity of the sample and is normally subtracted from the A260 value as a background reading for the calculation of DNA concentration, but excessive values may indicate non-specific contamination

 The most common purity calculation is determining the ratio of the absorbance at 260nm divided by the reading at 280nm.

Good-quality DNA will have an A260/A280 ratio of 1.7

2.0. A reading of 1.6 does not render the DNA unsuitable for any application, but lower ratios indicate more contaminants are present.

However, the best test of DNA quality is functionality in

the application of interest

 DNA concentration can be estimated by adjusting the A260 measurement for turbidity (measured by absorbance at A320), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50g/ml pure DNA. Concentration (g/ml) = (A260 reading A320 reading) dilution factor 50g/ml ,which is derived from Beers Law

Total yield is obtained by multiplying the DNA concentration by the final total purified sample volume.

DNA Yield (g) = DNA Concentration Total Sample Volume (ml)

PCR (Polymerase Chain Reaction):

Is a method whereby a specific sequence of nucleotides within a double-stranded DNA is amplified. Amplifies a region of DNA between two predetermined sites Oligonucleotides complementary to these ends of the DNA

serve as primers for synthesis of copies of the DNA between

the sites. Was invented by Kary Mullis and his colleagues in 1980s

PCR
The effect is that the new chains span the region delimited by the two chosen termini There are three phases in a single cycle of a PCR reaction: Denaturation at around 94C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle). Annealing at around 54C : Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached Extension at around 72C : The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template) The theoretical yield of amplified product is 2n where n is the number of cycles. More than a 109 -fold of the DNA stretch required may be obtained

Basic ingredients for PCR: The target DNA molecule to be amplified or the sample suspected to have the DNA (template). The dideoxynucleotides mix (dATP, dTTP, dCTP, dGTP ) The primers- about 20 nucleotides long and complementary to the ends of the target DNA Forward primers Reverse primers Thermo stable polymerase enzyme (Taq polymeraseisolated from Thermus aquaticus) Standard (ladder)(Reference) PCR buffer PCR grade water

PCR
100

Melting 94 oC Annealing Primers 50 oC Extension 72 oC

Melting 94 oC

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Advantage of PCR Very specific Fast Very important in diagnosing slow growing bacteria like Mycobacterium tuberculosis Sensitive DNA from one cell equivalent can be amplified PCR products can be directly sequenced PCR products can be directly cloned DNA sequences up to 30 kb can be amplified

Drawbacks of conventional PCR Time to run a gel to detect the amplicon Staining with ethidium bromide Difficulty to quantify Possibility of carry-over contamination

Controls for PCR

Blank reaction

Controls for contamination Contains all reagents except DNA template

Negative control reaction

Controls for specificity of the amplification reaction Contains all reagents & a DNA template lacking the target sequence

Positive control reaction

Controls for sensitivity Contains all reagents & a known target containing DNA template

PCR variations
Reverse transcriptase (RT)-PCR Nested PCR Multiplex PCR Inverse PCR Quantitative PCR Asymmetric PCR real time PCR

SDS-PAGE Gel Electrophoresis

Introduction Gel Electrophoresis is the study of the mobility of molecule

in an electric field.
Acryl amide and agarose are generally used for proteins

and DNA studies as a medium.

A protein will move with a net charge in an electric field (native charged or SDS-charged).

 The velocity of migration () of a protein depends on the

electric field strength (E), the net charge of the protein (z) and the frictional coefficient (f) Thus on a gel electrophoresis proteins will migrate as function of

1. Charge
2. Size (molecular weight) 3. Conformation

 Molecule small compared to the pore of the gel matrix

(medium) will move through the gel

whereas larger molecule will be almost immobile.

Intermediate size molecules will migrate with more or

less facility.

Gel Electrophoresis is used to (i) check purity of protein sample, (ii) calculate the protein size(SDS-PAGE), (iii) check protein conformation (native gel).

 The gel medium for protein gel is a mixture of acrylamide

and Bisacrylamide. Variation of the percentage of this mixture affects the size of

the pore of the matrix and therefore the migration of protein

samples. For small protein, a better resolution is obtained for high percentage of acrylamide / Bisacrylamide because it makes the pore tighter whereas for big proteins, low percentage of acrylamide / Bisacrylamide gives rise to a better resolution because the matrix is loose and allow migration of big molecule.

Native or non denaturating gel electrophoresis. Here the protein is migrating under native conditions which means that its functional structure is maintained and it will migrate according to size (molecular weight), charged and conformation. Under such conditions it is not possible to measure the protein molecular weight (size). No protein reference can be used because there are three parameters (size, charged and conformation) fixing the protein migration pattern. However, this technique is able to show difference in charged or conformation after for example, chemical modification or genetic mutation of the protein. To perform native gel electrophoresis, one need to know the pI (isoelectric point) of the protein since that will determine the net charged of the protein and tell you whether the protein will migrate towards anode or cathode.

SDS-PAGE or denaturing gel electrophoresis.

In order to use electrophoresis for protein molecular weight determination, it is necessary to eliminate at least two of the three parameters affecting the migration pattern. For this purpose, the protein sample is treated to have a uniform charge and its electrophoretic mobility become size dependent only.

 This is possible by treating the protein under denaturating conditions, meaning that secondary, tertiary and quaternary structures are disrupted to produce a linear polypeptide

chain coated with negatively charged SDS molecules.

Then the protein migrates only depending on its size (molecular weight) Because the number of SDS molecule bound to a protein is proportional to the number of amino acids.

 1.4g of SDS binds to 1 g of protein which means that there

is one SDS molecule per every 2 aminoacids. Because SDS is negatively charged, protein will migrate towards the positive electrode.

Hybridization Tests
Principles of hybridization:

Hybridization is based on the principle that nucleic acid strands with complementary sequences bind to each other under the appropriate conditions. Final detection will be by using radioactive elements to aid in the visualization. The most used 32P Northern blotting (for RNA) & southern blotting (for DNA) follow the same principles. Western blotting (for protein) also follow the same principles except that in place of probes labeled antibodies are used.

Northern Blotting
Northern blot hybridization is a technique used to detect and quantify a given mRNA species within a mixture of

RNAs in vitro.
Steps in Northern blotting: The RNAs in the mixture are separated according to size by electrophoresis in an agarose gel. The RNAs are transferred from the gel to a membrane filter. This transfer step is also called blotting

 The target mRNA is detected by incubating the membrane

with a specific, radiolabeled nucleic acid probe. The probe anneals by base pairing with the specific mRNA species of interest. The membrane serves as the solid support that allows separation of specifically bound probe from excess probe, which is simply washed away. Probe that is bound to RNA on the membrane is then detected by autoradiography.

The probe has three essential features: It consists of a sequence complementary to the mRNA of interest.

It is single-stranded.

It contains a radioactive label so that it is readily detected. (Oligonucleotide probes are often labeled by including a radioactive base during their synthesis). To accurately quantitative the mRNA species of interest, the mRNA, not the probe, must be the limiting entity. Therefore, the amount of probe must exceed the amount of mRNA on the filter.

Applications of Northern blotting is mainly in basic research to: Detect multiple forms of an mRNA derived from a single gene (such as precursors and products during processing or alternative mRNAs produced by differential splicing). Monitor changes in the expression of a gene during different stages of growth or development.

Detect differences in gene expression in different cell types.

Compare the amounts and sizes of mRNAs of genes involved in various cellular processes (e.g., cell cycle regulation) in different types of organisms.

Southern blotting (named after its inventor Edward Southern): A cloned gene (or part of a gene) or a PCR fragment can be used as a probe for finding segments of (genomic) DNA that have the same or a very similar sequence.
The genomic DNA is extracted and digested with a restriction enzyme that will cut it into defined populations of segments of specific size. These fragments can be separated into groups of fragments of the same length by using (agarose) gel electrophoresis.
DNA is negatively charged and will migrate toward the positive pole. The electric field causes the molecules to move through the pores of the gel in a wormlike fashion at speeds inversely proportional to their size:

 After fractionation, the gel is soaked in alkali to denature the double-stranded DNA and then blotted onto a piece of porous positively charged membrane, where they stay in the same relative positions.
After having been soaked in alkali to separate the DNA strands and link the DNA to the membrane, the membrane is placed in a hybridization buffer containing the probe. The single-stranded probe will find and bind to its complementary DNA sequence.

 Unbound probe is removed by washing steps. The probe must be labeled such that it can be readily located on the membrane once it has bound to its complementary target sequence. Probes can be labeled with radioactive atoms (for example by adding a 32P-atom at the 5'-end of DNA by the enzyme polynucleotide kinase and (32P-ATP as substrate, or by incorporation of a labeled radioactive or fluorescent precursor during in vitro DNA synthesis). Radioactive probes can be detected by autoradiography (Xray sensitive film) or by photomultipliers that emit light in response to the excitation by the beta particles emitted from 32P, 33P and 35S. Fluorescent probes can be detected by irradiation with appropriate wavelength UV light and monitoring the longer wavelength that is emitted in response.

Western Blotting
The same in principle with Northern or Southern blotting but it is for proteins.

Electrophoresing proteins
Blotting to a membrane & reacting with a specific

antibody
The antibody is detected with a labeled secondary antibody.

Dot and slot blot hybridization

Often it is informative to quantify the abundance of a certain RNA or DNA in the extracted nucleic acid mixture by dot

blot or slot blot hybridization without prior digestion and

electrophoresis. In the procedure, the nucleic acid mixture is blotted to a membrane where the hybridization is carried out. The difference between dot and slot blot procedures is in

the way that the nucleic acid mixture is blotted onto the
membranrane

 In dot blotting the nucleic acids are blotted as circular blots, whereas in slot blotting they are blotted into rectangular slots. The latter method allows a more precise observation of different hybridization signal intensities. Quantification of a certain RNA/DNA compared with total RNA/DNA can be obtained by hybridization with universal and specific oligonucleotide probes. The relative abundance is calculated by dividing the amount of specific probe bound to a given sample by the amount of hybridized universal probe measured e.g. as fluorescence intensity (fluorescent probes) or counts per minute (radioactively labelled probes).

FISH (fluorescent in situ hybridization)

In the FISH technique, a DNA or RNA probe is labeled by attaching a fluorescent dye and used to hybridize to a complementary nucleic acid from a mixture. The binding of a fluorescent probe to its target can be seen microscopically. By treating cells with the appropriate reagents, membranes become permeable and allow the penetration of the probe. FISH can be applied directly to cells in culture or in the natural environment and offers therefore a rapid method for assessing the composition of microbial communities directly by microscopy.

Microarrays
A DNA microarray is a multiplex technology used in molecular biology.

It consists of an arrayed series of thousands of microscopic

spots of DNA oligunuculeotide, called features, each containing picomoles (1012 moles) of a specific DNA sequence, known as probes (or reporters).

 These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.
Since an array can contain tens of thousands of probes, a microarray experiment can accomplish many genetic tests in parallel. Therefore arrays have dramatically accelerated many types of investigation

 The core principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.
A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off of non-specific bonding sequences, only strongly paired strands will remain hybridized.

 So fluorescently labelled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases, the hybridization conditions (such as temperature), and washing after hybridization.
Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position.

 DNA microarrays can be used to measure changes in

expression levels, to detect single nucleotide

polymorphism (SNPs), or to genotype or resequence mutant genomes

DNA sequencing
Basically, there are three applications of sequencing: sequencing of short regions of DNA to identify mutations

of interest or single nucleotide polymorphisms (SNPs);

sequencing of complete genes and associated upstream and downstream control regions; sequencing of complete genomes. the clone-by-clone approach and whole-genome shotgun sequencing. DNA sequencing is the determination of the precise sequence of nucleotides along a DNA strand

DNA sequencing (Sanger method)

The underlying principle of DNA sequencing is based on the separation by size of nested sets of DNA molecules that differ in length by one nucleotide only. Each of the DNA fragments that are generated start at a common 5'-end and terminate at one of several alternative 3'-ends. Members of a given set have a particular type of base at their 3'-end: Four sets are generated, one terminating with G, one with A, one with T and one with C.

 This method uses chain-terminating nucleotides in an in vitro enzymatic DNA synthesis approach.
Chain terminating nucleotides (2'-, 3'- dideoxy derivatives, ddNTPs) are derivatives of the 2'deoxynucleoside triphosphates that lack the OHgroup at the 3' position. Therefore, when such a nucleotide is incorporated there is no free 3'-OH end and the chain can not be extended. Thus, DNA synthesis will stop.

General procedure: Four different DNA synthesis reactions are made, each one will contain the four normal dNTPs and a small amount of one ddNTP (about 100-fold less than the corresponding dNTP). This will force DNA synthesis to abort at a frequency of one in one hundred every time the DNA polymerase encounters a nucleotide complementary to the corresponding ddNTP derivative in the template DNA. Because all of the DNA chains start from the same point (the 5'-end of the oligonucleotide used in the enzymatic DNA synthesis reaction), the chainterminating reactions will generate a nested set of fragments, all sharing the same 5'-end but differing in length and hence their 3'-ends.

 The comparison of all four lanes (four different reactions with a different ddNTP) allows to establish the sequence of the DNA segment.

If the fragments are labeled by incorporation of radioactive

nucleotides (32P, 33P or 35S derivatives), the fragments can be separated by acrylamide gel electrophoresis and detected by

autoradiography.
Radioactive labeling: ddNTPs or one of the dNTPs or The primer

 If fluorescent labeled derivatives are used, column chromatography is used to separate the fragments and the read out is given by laser detectors. This is now generally performed with automated sequencing machines, sequenators, that can have up to 384 fractionation columns that can generate over 200,000 nucleotides of raw DNA sequence in a few hours.

 Labeling is done by DNA polymerase on dideoxy nucleotide & this tag accomplishes two operations in one step It terminates the synthesis It attaches the fluorophore to the end of the molecule Alternatively, fluorescent primers can be used with nonlabeled dideoxynucleotides (using four different fluorescent dyes). The DNA bands are detected when fluoresce as they pass a detector that recognize the four different colors as four different nucleotides. Note: Sanger sequencing has an accurate read length of 500800 bases

Pyrosequencing
Permits sequence analysis in real time Pyrosequencing is a DNA sequencing method that involves determining which of the four bases is incorporated at each step in the copying of a DNA template. As DNA polymerase moves along a single stranded template, each of the four nucleoside triphosphates is fed sequentially and then removed.

If one of the four bases is incorporated then pyrophosphate is released and this is detected in an enzyme cascade that emits light.

(Oligonucleotide)n + nucleotide

Polymerase

(Oligonucleotide)n + 1 + PPi ATP + sulfate

PPi + APS

ATP Sulfurylase Luciferase

ATP + luciferin + O2

AMP + PPi + oxyluciferin + LIGHT

The general principle of pyrosequencing: A polymerase catalyzes incorporation of nucleotides into a nucleic acid chain. As each nucleotide is incorporated a pyrophosphate (PPi) molecule is released and incorporated into ATP by ATP sulfurylase. On addition of luciferin and the enzyme luciferase, this ATP is degraded to AMP with the production of light.

There are two variants of the pyrosequencing technique: In solid-phase pyrosequencing (Ronaghi et al. 1996), the DNA to be sequenced is immobilized and a washing step is used to remove the excess substrate after each nucleotide addition. In liquid-phase sequencing (Ronaghi et al. 1998b) a nucleotide degrading enzyme (apyrase) is introduced to make a four-enzyme system. Addition of this enzyme has eliminated the need for a solid support and intermediate washing thereby enabling the pyrosequencing reaction to be performed in a single tube.

Genome sequencing
sequencing of complete genomes can be done by two approach:

the clone-by-clone approach

and whole-genome shotgun sequencing. Used by human genome sequencing project

DNA fingerprinting (DNA typing or DNA profiling)

Traditionally, one of the most accurate methods for placing an individual at the scene of a crime has been a fingerprint. With the advent of recombinant DNA technology, a more powerful tool is now available: DNA fingerprinting (also called DNA typing or DNA profiling). DNA fingerprinting is based on sequence polymorphisms, slight sequence differences (usually single base-pair changes) between individuals, 1 bp in every 1,000 bp, on average.

 Each difference from the prototype human genome sequence (the first one obtained) occurs in some fraction of the human population; every individual has some differences. Some of the sequence changes affect recognition sites for restriction enzymes, resulting in variation in the size of DNA fragments produced by digestion with a particular restriction enzyme. These variations are restrictionfragment length polymorphisms (RFLPs).

The detection of RFLPs relies on a specialized hybridization procedure called Southern blotting

Procedure : DNA fragments from digestion of genomic DNA by restriction endonucleases are separated by size electrophoretically, denatured by soaking the agarose gel in alkali, and then blotted onto a nylon membrane to reproduce the distribution of fragments in the gel. The membrane is immersed in a solution containing a radioactively labeled DNA probe. A probe for a sequence that is repeated several times in the human genome generally identifies a few of the thousands of DNA fragments generated when the human genome is digested with a restriction endonuclease. Autoradiography reveals the fragments to which the probe hybridizes,

 The genomic DNA sequences used in these tests are generally regions containing repetitive DNA The number of repeated units in these DNA regions varies

among individuals (except between identical twins).

With a suitable probe, the pattern of bands produced by DNA fingerprinting is distinctive for each individual.

Combining the use of several probes makes the test so

selective that it can positively identify a single individual in the entire human population. RFLP analysis sensitivity is augmented by using PCR to amplify vanishingly small amounts of DNA

Suspect 2

reference

reference

Suspect 1

Fig. The Southern blot procedure, as applied to DNA fingerprinting: One suspects (suspect 1) DNA exhibits a banding pattern identical to that of a semen sample taken from the victim(evidence).

reference reference

Evidence

Victim

Summary
Since DNA is inside the cell, cells envelop must be broken
down to release their contents using lysing buffers

Cells containing the desired plasmid or cosmid are harvested

by centrifugation, incubated in a lysozyme buffer, and treated

with alkaline detergent that breaks the cells, and the DNA
and proteins- are released into the medium.

PCR is a method for amplifying a specific sequence of DNA

from a complex mixture.

 Gel electrophoresis refers to the migration of all charged

solutes in a medium under the influence of electrical field Hybridization is based on the principle that nucleic acid strands with complementary sequences bind to each other under the appropriate conditions. DNA sequencing is the process of determining the order of the nucleotides along a DNA strand. When human DNA is digested with restriction enzyme, a

polymorphic locus yield restriction fragment of different sizes

called RFLPs (restriction fragment length polymorphism)

Review question
1. Describe extraction, purification of DNA and quantification of DNA yield

2. Describe gel electrophoresis and list down factors that

affect it 3. List and explain hybridization tests 4. explain DNA sequencing methods 5. Describe genome sequencing

References
Robert F. weaver, Philip W. Hedrick. Genetics. Darnel, Lodish, Baltimore. Molecular Cell Biology James D. Watson: Recombinant DNA Robert F. Weaver. Molecular biology Richard J. Epistein: Human Molecular Biology P.K. Gupta: Cell and Molecular Biology

Tarek H. EL-Metwally. Basic Medical Molecular

Biology: A Comprehensive update .