Information

Why re-label the strand ends in 3' DNA labelling?


I have a problem with a molecular biology question; I don't understand how DNA 3' labelling works. I took a diagram from my lesson and tried to understand with it; this is what I understood. If I'm wrong, please tell me and correct me.

  • We take a restriction enzyme which will cut both strands. The bottom strand will be a 5' end. So, the top strand is shorter than the bottom strand.

  • We will add a Klenow fragment which, with its polymerase activity, polymerizes in the 5'->3' direction the missing part of the top strand, with dNTP α 32P (labelled by a radioactive phosphate in the alpha position).

  • We get two strands which have the same size, of which one will be strongly labelled.

  • We finish with a terminal transferase which will catalyse the addition of dNTP α 32P on the 3'OH ends of both DNA strands (in order to label these ends).

But, if I'm right, could you explain the necessity of the last step; why label both ends since we have already labelled the top strand?


There are many ways to label nucleic acids for probes (Summary of Methods).

While there are cases where Klenow fragment-mediated fill-in should sufficiently label both strands of your probe, there are many others where this would not be enough. Here are two illustrative, although somewhat contrived, examples:

Example 1: Double digest with 5' and 3' overhangs
Imagine you did a double digest with BamHI and KpnI to release your probe sequence from the plasmid in which you propagate the sequence. BamHI will give you a 5' overhang, but KpnI gives a 3' overhang.

Resulting BamHI & KpnI double digest fragment: 5'- GATCCNNNNNNNG - 3' ||||||||| 3'- GNNNNNNNCCATG - 5'

Subsequent treatment with the Klenow fragment + $alpha$P32-dATP + cold dNTPs will both fill-in the 5'-overhang (Klenow's 5'->3' polymerase activity) and remove the 3' overhang (Klenow's 3'->5' exonuclease activity).

Post-Klenow reaction (bold = new bases, * = radio-labeled nucleotide): 5'- GATCCNNNNNNNG - 3' ||||||||||||| 3'- CTAGGNNNNNNNC - 5' *

At this point only one strand is radio-labeled so the terminal transferase step is needed to add a label to the top strand.

When using $alpha$P32-dNTPs, you often pick only one base to be radioactive (eg dATP as above). Sometimes you also just spike the reaction with radioactive dNTP leaving a population of cold dNTPs to be used most of the time. In either case you now have the chance of not incorporating only non-radioactive bases.

Example 2: Fill-in reaction that fails to incorporate $alpha$P32-dATP:

Imagine a digest with PspI and want to fill-in the 5' overhangs using $alpha$P32-dATP + cold dNTPs. PspI recognizes CCWGG sequences where W is either A or T so that some fill-ins will incorporate the $alpha$P32-dATP, but others will not.

Possible digests and fill-in reactions: 5'- CCTGGNNNNNNNNNNCCTGG - 3' |||||||||||||||||||| 3'- GGACCNNNNNNNNNNGGACC - 5' | Digest with PspI | V 5'- CCTGGNNNNNNNNNN - 3' |||||||||| 3'- NNNNNNNNNNGGACC - 5' | Fill-in with Klenow + hot dATP + cold dNTPs | V 5'- CCTGGNNNNNNNNNNCCTGG - 3' |||||||||||||||||||| 3'- GGACCNNNNNNNNNNGGACC - 5' *

Again you are left with only one strand being labeled (or no strand labeling if the sequence was 5'- CCAGGNNNNNCCTGG - 3') and a need for the terminal transferase step.


Why is there a leading and lagging strand during DNA replication

The double stranded DNA is first unzipped by an enzyme called helicase. Helicase separates the two strands of DNA, and creates the replication fork. Essentialy two single stranded DNA chains are created, both facing different directions. On the leading strand, an RNA primer is created by RNA ploymerase and DNA polymerase III will continously build that strand, since it is building the DNA chain in the same direction as helicase unzips the DNA. On the lagging strand, the DNA plymerase moves the opposite direction as helicase, thus it can only copy a small length of DNA at one time. Because of the different directions the two enzymes moves on the lagging strand, the DNA chain is only synthetised in small fragments. Hence it is called the lagging strand.


Question : 3. Given the following piece of DNA, draw the complementary strand. Label 5' and 3" ends. 5'- ATG CTACCCTGCATG CATTCCC-3' 4. Write out each of the single strands of the DNA sequence you have above. On each strand, draw a 5-nucleotide RNA primer in the proper place. Label the 5' and 3' ends TA TA 7 5]braw both of the double stranded DNA molecules after

Not sure how to do number 5


DNA: Restriction Mapping and Nucleotide Sequencing

Read this article to learn about the restriction mapping of DNA which involves the size analysis of restriction fragments and also learn about nucleotide sequencing of DNA for which two techniques have been developed:

(1) based on enzymatic method called Sanger sequencing and (2) based on chemical method called Maxam and Gilbert sequencing.

Restriction Mapping of DNA Fragments:

This involves the size analysis of restriction fragments produced by several restriction enzymes individually and in combination. For example, in Fig. 3.2 restriction sites of two enzymes A and B are being mapped. Cleavage with A gives fragments 2 and 7 kilo bases from a 9 kb molecule, hence we can have position of single A site from one end.

Similarly, B gives fragments 3 kb and 6 kb away, so it has a single site 3 kb from one end but it is still not clear whether this site is near A’s site or is at opposite end of DNA. This can be resolved by double digestion. If the resultant fragments are 2 kb, 3 kb and 4 kb away, then A and B cut at opposite ends of the molecule if they are 1 kb, 2 kb and 6 kb away the sites are near to each other. It is worth stating here that the mapping of real molecules is rarely as simple as this.

Nucleotide Sequencing of DNA:

The precise usage of codons, information regarding mutations and polymorphisms and identifica­tion of gene regulatory control sequences can only be elucidated by analyzing DNA sequences. Two techniques have been developed for this, one based on an enzymic method frequently termed Sanger sequencing and chemical method called Maxam and Gilbert sequencing.

Sanger’s Sequencing or Dideoxynucelotide Chain Terminators:

In this the reaction mixture is divided into four groups, representing the four dNTPs A, C, G and T. In addition to all the dNTPs being present in the A tube an analogue of dATP is added (2′, 3′ ddATP) is added that is similar to A but has no 3′ hydroxyl group and so will terminate the growing chain. Situation for other tubes ddC, ddG and ddT are identical except they contain ddCTP, ddGTP and ddTTp respectively.

Since the incorporation of ddNTP rather than dNTP is a random event, the reaction will produce new molecule varying widely in length, but all terminating at the same type of base. Thus four sets of DNA sequences are generated, each terminating at a different type of base, but all have a common 5′- end. The four labelled and chain-terminated samples are then denatured by heating and loaded next to each other for electrophoresis.

Electrophoresis is performed at 70°C in pres­ence of urea, to prevent renaturation of DNA. Very thin and long gels are used for maximum resolution over a wide range of fragment lengths. After electrophoresis, the position of radioactive DNA bands on the gel is determined by autoradiography.

Since every band in the track from dideoxyadenosine triphosphate must contain molecules that terminate at adenine, and those in ddCTP terminate at cytosine, etc., it is possible to read the sequence of the newly synthesized strand from autoradiograph, provided that the gel can resolve differences in length equal to single nucleotide. Under ideal conditions, sequences up to about 400 bases length can be read from one gel.

Direct PCR Sequencing:

It is possible to undertake nucleotide sequencing from double-stranded molecules such as plasmid cloning vectors and PCR products but the double-stranded DNA must be denatured prior to annealing with primer. In case of plasmids, an alkaline denaturation step is sufficient. However, for PCR products this is more problematic and a focus of much research. Unlike plasmids, PCR products are short and re-annealed rapidly, so preventing the re-annealing process or biasing the amplification towards one strand by using a primer ratio of 100:1 can overcome this problem to a certain extent.

Denaturants such as form amide or dimethylsulphooxide (DMSO) are usually em­ployed to prevent renaturation of PCR strands after their separation it is possible to physically separate and retain one PCR strands by incorporating a molecule such as biotin into one of the primer, which can be recover after PCR by affinity chromatography with streptavidin, leaving the complimentary PCR strand. Thus it provided high quality single stranded DNA for sequencing.

One of the most useful methods of sequencing PCR products is termed PCR cycle sequencing. This is not strictly a PCR, since it involves linear amplification with a single primer in about 20 PCR cycles. Radiolabeled or fluores­cent—labelled dideoxynucleotides are then introduced into the final stages of reaction to generate chain termination extension products. Automated direct PCR sequencing is increasingly being refined, allowing greater lengths of DNA to be analysed in one sequencing run.

Automated Fluorescent DNA Sequencing:

This involves dideoxynucleotides labelled with different flurochromes and are used to carry out chain termination as in standard re­actions. The advantage of this modification is that, since different labellel is incorporated in each ddNTP all the products are run on same denaturing electrophoresis gel.

Each product with their base specific dye is excited by a laser and dye then emits light at its characteristic wavelength. A diffraction grating separates the emissions, which are detected by charge couple device (CCD) and the sequence is interpreted by computer. In addition to real time sequencing, the length of the sequence that may be analysed is in excess of 500 bp.

Maxam and Gilbert Sequencing:

This is a chemical method of sequencing developed by Maxam and Gilbert and the method is often used for sequencing of small fragments of DNA such as oligonucleotides. A radioactive label is added to either the 3′ or the 5′ end of a double stranded DNA preparation. The strands are then separated by electrophoresis under denaturating conditions and analysed separately.

DNA labelled at one end is divided into four aliquots each is treated with chemicals that act on specific bases by methylation or removal of bases. Conditions are chosen such that each molecule is modified at only one position along its length and every base in the DNA strand has equal chances of being modi­fied.

After modification reactions separate samples are cleaved by piperidine, which breaks phosphodiester bonds exclusively at the 5′-side of nucleotides whose base has been modified. The result is similar to that produced by Sanger method, since each sample now contains radiolabelled molecules of various length, all with one end common (the labelled end), and with the other end cut at the same type of base. Analysis of the reaction products by electrophoresis is as already de­scribed for Sanger method.


DNA Labeling

Nucleic acids are readily labeled with tags that facilitate detection or purification. A variety of enzymatic or chemical methods are available to generate nucleic acids labeled with radioactive phosphates, fluorophores, or nucleotides modified with biotin or digoxygenin for example.

Nucleic acids may be labeled at their 5´ end, their 3´ end, or throughout the molecule depending on the application. For hybridization reactions (Southern or northern blotting) it is usually advantageous to generate high specific activity probes with label distributed throughout the nucleic acid, through techniques such as nick translation, random priming, by PCR or in vitro transcription using labeled dNTPs or NTPs. For applications involving protein interactions, such as gel-shift assays or pull-downs, it is generally beneficial to generate end-labeled probes to prevent steric interference of the interaction. This can be achieved with end-labeling protocols or with PCR using primers bearing the required modification.

New England Biolabs offers a number of reagents and kits suitable for labeling single-stranded or double-stranded DNA and RNA at either the molecule ends or randomly throughout the nucleic acid.

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Transcriptional Labeling of RNA Probes

For some applications, DIG-labeled RNA is a more effective hybridization probe than DIG-labeled DNA. For example, DIG-labeled RNA probes can detect rare mRNAs in nanogram amounts of total RNA. These labeled RNA probes are generated by in vitro transcription from a DNA template. In the RNA transcription method, DNA is cloned into the multiple cloning site of a transcription vector between promoters for different RNA polymerases (such as T7, SP6, or T3 RNA polymerase). The template is then linearized by cleavage of the vector at a unique site (near the insert). An RNA polymerase transcribes the insert DNA into an antisense RNA copy in the presence of a mixture of ribonucleotides (including DIG-UTP). During the reaction, the DNA can be transcribed many times (up to a hundredfold) to generate a large amount of full-length DIG-labeled RNA copies (10-20 μg RNA from 1 μg DNA in a standard reaction). DIG is incorporated into the RNA at approximately every 25-30 nucleotides.

Reaction principle

The DNA template to be transcribed is cloned into the polylinker site of an appropriate transcription vector, which contains promoters for SP6 and or T3 and T7 RNA polymerases. After linearization at a suitable site, RNA is transcribed in the presence of DIG-11-UTP. Under standard conditions, approximately 10 μg of full-length DIG-labeled RNA are transcribed from 1 μg of template.

The following tips are critical for successful RNA probe labeling:

RNases

RNases are ubiquitous and do not require any cofactors for activity. If you want to be successful, take all possible precautions to prevent RNase contamination. For instance:

  • It is recommended to use disposable plasticware, oven-baked glassware, or plasticware that has been decontaminated with RNase ZAP or similar reagents.
  • Prepare all solutions with water that has been treated with diethyl-pyrocarbonate (DEPC) or dimethyldicarbonate (DMDC) and autoclave the solutions.
  • Wear gloves throughout the procedure.
  • Labeling efficiency depends greatly on the purity of the DNA template. Template should be highly purified.
  • The final template must be linearized, phenol/chloroform extracted, and ethanol precipitated.

Template sequence

  • Some primer and/or polylinker regions in DNA templates are homologous to portions of the ribosomal 28s and 18s RNA sequences. Therefore, labeled probes may generate specific, but unwanted signals in samples that contain these prominent RNAs. To minimize this effect, remove as much of the polylinker sequences from your template as possible.
  • If you use PCR to make the DNA template, the product of the Expand High Fidelity reaction contains some fragments with a single 3´ A overhang. This overhang may produce wraparound products in the transcriptional labeling reaction.

Template length

  • Optimal template length is approximately 1 kb
  • Minimum length should be at least 200 bp

Storage of probe

  • For long term stability, RNA probes should be aliquoted and stored at -20 °C or -70 °C
  • DIG-labeled RNA probes are stable for at least 1 year at -20 °C or -70 °C in ethanol

Probe sensitivity

  • To quickly determine the sensitivity of a DIG-labeled antisense RNA probe, prepare the corresponding sense RNA (unlabeled) by in vitro transcription. Then use the purified sense transcript at varying concentrations as target on a northern blot. From the result of the blot you can easily determine the lowest amount of target (sense transcript) that can be detected by labeled probe (antisense transcript).

How probe hybridization occurs?

Traditionally, the method is known as southern blot (for DNA hybridization) or northern blot (for RNA hybridization).

In the very first step, the DNA sequence of our interest is digested with the restriction endonuclease and allow to immobilised on the nitrocellulose paper.

A complementary probe is selected, labelled and denatured first.

Then in the next step, the probe mixture allows binding on the nitrocellulose paper, if it finds the exact complementary sequence, it will bind.

The unbound probes are removed by washing the nitrocellulose paper with the wash buffer.

The final results are collected using the autoradiography method.

In the modern version of this technique- called a microarray, the probes are hybridized instead of sample DNA.

The sample is allowed to hybridize on the surface containing millions of probe, thus microarray enables to screen many mutations or alterations at once.


DNA and RNA (With Diagram)

Watson and Crick (1953) have proposed a model for the structure of DNA molecule which is now usually accepted by all. According to this model called as Watson Crick Model, the DNA molecule is a double he­lix structure consisting of two long polynucleotide chains coiled round each other around an imaginary axis and running opposite to each other. (Fig. 9.14).

Each polynucleotide chain consists of thousands of nucleotide units.

The back-bone of the two helices of polynucleotide chain consists of deoxyribose phosphates while the bases are present on the inner sides.

The bases of the one polynucleotide chain are complementary to the bases of the other polynucleotide chain and are joined together by hydrogen bonds (Fig. 9.16).

The base pairing is very specific (Fig 9.15), The complementary bases are:-

The ratio of purine and pyrimidine bases is 1: 1.

The distance between two subsequent base pairs in the polynucleotide chain is 3.4 Å.

Each turn of the two polynucleotide chains is completed after 10 base pairs i.e., a distance of 34 Å.

The distance between the axis and the sugar phosphate region is about 10 Å. The helical coiling is right handed.

(1. D.N.A. molecules are gigantic. Their molecular weights are in the region of several millions. 2. In some bacteriophages i.e., the viruses that attack bacteria, the DNA is single stranded.)

There are different forms of DNA in living organisms viz., A, B, C, and rarely D and E. In all these forms, the helical coiling of DNA molecule is right handed. These forms differ in number of base pairs (bp) per turn, diameter of the helix and similar other minor details. Most common and usual form of DNA found in living organisms is B form which is called as B-DNA. The preceding account of the structure of DNA in-fact pertains to the B-DNA.

Recently, another form of DNA has artificially been synthesized in which helical coiling of DNA is left handed and the phosphate backbone of the polynucleotides follows a zig-zag course. This form of DNA has therefore, been designated as Z-DNA. Some of the other impor­tant features of Z-DNA one: 1. Each turn of the two polynucleotide chains is completed after 12 base pairs i.e., a distance of about 45 Å. 2. Distance between two subsequent base pairs is 3.7 Å. 3.

The distance between axis and sugar-phosphate is 9 Å. 4. Alternate deoxy-ribose sugar units in the polynucleotide chain have inverse orientation (one such unit having ethe­real oxygen facing upward and the other subsequent unit facing downward).

Ribose Nucleic Acid (RNA):

RNA is a single stranded structure consisting of only one polynucleotide chain. If some­time the complementary bases come very close to each other, hydrogen bonds are established between them to give polynucleotide chain a helical appearance like DNA.

RNA consists of the following bases:

The ratio of purine and pyrimidine bases is not 1:1.

The pentose sugar is β-D-Ribose.

The size of RNA molecule is very small in comparison to the DNA molecule. Molecular wt. of RNA may range from several thousands to some lakhs.

There are 3 different forms of RNAs in plant cells:

Molecular wt. of m-RNA is higher among different types of RNAs usually varying from 5- 10 lakhs. These constitute 5-10% of the total RNA is the cell.

m-RNA is synthesized in nucleolus and after taking genetic information from DNA goes into the cytoplasm and helps in the formation, of specific protein. m-RNA are short lived.

Sequence of 3 bases of nucleotides in m-RNA molecule constitutes a codon. Actually the genetic information obtained from the DNA is encoded in codons which are specific (for de­tails see genetic code).

(ii) Ribosomal RNA (r-RNA):

r-RNA is found in ribosome which act as template for the synthesis of proteins. It is of most stable kind and constitutes about 80% of the total RNA in the cell.

(iii) Transfer or Soluble RNA (t-RNA or s-RNA):

The structures of many t-RNA molecules are known in quite detail. These are compara­tively very small with a molecular weight of about 25000. Basic structure of all t-RNA mol­ecules is on the clover leaf pattern. Clover leaf model of t-RNA is given in Fig. 9.17.

t-RNAs are found in cytoplasm and consist of only about 80 bases. These constitute 10- 15% of total RNA in the cell.

t-RNAs contain many unusual bases and nucleotides. These are e.g., pseudouridine (Ψ), dihydrouridine (DHU), inosine (I) etc. Methylation of bases is also common.

All t-RNA molecules contain Guanine (G) at 5′ end. The 3′ end always ends in the base sequence Cytosine-Cytosine-Adenine (CCA). During protein synthesis this end in fact picks up the amino acid and transfers it to the growing polypeptide chain and hence these RNAs are called as t-RNAs or transfer RNAs.

These t-RNAs are also called as s-RNA or soluble RNAs because they are soluble in IM NaCl.

t-RNA molecules are folded in a clover leaf pattern with three or more double helical re­gions (like DNA) terminating in loops.

Three important loops of t-RNA are:

(ii) Amino acyl synthetase binding loop

(iii) Ribosomal binding loop.

Anticodon loop consists of 7 bases. At the free end, 3 unpaired bases constitute the anticodon which is complementary to codon in m-RNA. Aminoacyl synthetase binding loop consists 8-12 bases. Because of the presence of dihydrouridines in this loop, it is also known as DHU loop.

The ribosomal binding loop consists of 7 bases. In contains sequence of GTΨC and hence is also called as GTΨC loop.

There are different t-RNA molecules with specific anticodons to pick up specific amino acids. However, many t-RNAs may be specific to a particular amino acid or a single t-RNA species may recognise several amino acids.

The t-RNA molecule whose structure was first given by Holley in detail is Yeast alanyl- t-RNA (Fig. 9.18).


Nucleic acids 7.1 HL

The basics of DNA structure and DNA replication were covered in the SL topic so this topic looks at some extra details including, the 3' and 5' ends of the two antiparallel strands and further details about the enzymes responsible for DNA replication and some of the DNA regions which don't code for proteins but that regulate gene expression, form introns, telomeres or genes for tRNA. To understand how modern sequencing machines work a clear understanding of PCR and gel electrophoresis is also required.

Key concepts

Learn and test your biological vocabulary for 7.1 Nucleic acids using these flashcards.

Essentials - quick revision through the whole topic

These slides summarise the essential understanding and skills in this topic.
They contain short explanations in text and images - good revision for all students.

Read the slides and look up any words or details you find difficult to understand.

Exam style question

How to prepare to answer a difficult IB question about DNA replication.
From your revision notes create between 8 and 10 quick questions.
Test yourself. Can you remember the answers?

1. What does helicase do?
2. Which proteins keep the DNA strands separate?
3. What is the role of the enzyme primase?
4. How is DNA polymerase III different from primase?
5. Which direction does DNA polymerase III along the template stand?
6. What is the leading strand?
7. What is the lagging strand?
8. Why are okazaki fragments produced on the lagging strand?
9. What is the role of the enzyme DNA polymerase I ?
10. What is the role of DNA ligase enzyme?

Separates the two DNA strands by breaking hydrogen bonds.

2. Which proteins keep the DNA strands separate?

Single strand binding proteins.

3. What is the role of the enzyme primase?

Primase attaches an RNA primer to the DNA strand.

4. How is DNA polymerase III different from primase?

DNA polymerase III adds DNA nucleotides, Primase adds RNA nucleotides to the strand.

5. Which direction does DNA polymerase III along the template stand?

6. What is the leading strand?

The strand that DNA polymerase III can add nucleotides continuously to.

7. What is the lagging strand?

The strand on which DNA polymerase III can only work in short sections.

8. Why are okazaki fragments produced on the lagging strand?

DNA polymerase III cannot produce a new strand continuously.

9. What is the role of the enzyme DNA polymerase I ?

DNA polymerase I replace the RNA nucleotides of the primer with DNA nucleotides.

10. What is the role of DNA ligase enzyme?

DNA ligase joins okazaki fragments together.

Summary list for topic 7.3 Translation

  • Part of DNA supercoiling are structures called Nucleosomes.
  • DNA structure gives a clue to the mechanism of DNA replication.
  • Non-coding regions of DNA have other important functions, limited to regulators of gene expression, introns, telomeres and genes for tRNAs.

DNA replication (in prokaryotes only)

  • DNA polymerase enzymes can only add nucleotides to the 3&rsquo end of a primer.
  • Continuous DNA replication occurs on the leading strand and discontinuous on the lagging strand.
  • A complex group of enzymes do DNA replication including helicase, DNA gyrase, single strand binding proteins, DNA primase and DNA polymerases I and III.
  • DNA replication makes a second chromatid in each chromosome in interphase before meiosis.
  • Crossing over exchanges pieces of DNA between non-sister homologous chromatids and forms new combinations of alleles on the chromosomes formed in meiosis.
  • See that Rosalind Franklin&rsquos and Maurice Wilkins&rsquo X-ray diffraction work gave evidence for helix and two strands in DNA structure.
  • Awareness that the Sanger method of base sequencing uses nucleotides containing dideoxyribonucleic acid (DNA with deoxyribose missing 2 oxygen molecules) to stop DNA replication at a specific base which allows sequencing using fluorescent markers and computers. (Sanger chain termination. Video here).
  • Awareness that in DNA profiling Tandem repeats are used as these vary greatly from person to person.
  • Ability to analyse of results of the Hershey and Chase experiment providing evidence that DNA is the genetic material.(This is a nice graphic).
  • Analyse of molecular visualisations of the association between protein and DNA in a nucleosome.

Mindmaps

These diagram summaries cover the main details of topic 3.5 Genetic modification.
Study them and draw your own list or concept map, from memory if you can.

Test yourself - multiple choice questions

This quiz contains multiple choice questions covering the understandings and skills in this topic.

7.1 DNA structure and replication 1 / 1

It is estimated that a large percentage of human DNA is 'non-coding'.

Which of the following answers lists important functions of these non-coding regions?

Recessive alleles and genes for tRNA molecules

Regulators of gene expression, and genes for DNA replication enzymes

Exons and genes for tRNA molecules

Non-coding regions of DNA have other important functions, limited to regulators of gene expression, introns, telomeres and genes for tRNAs.

Which of the following is a role of nucleosomes in eukaryotes?

Part of supercoiling of DNA.

Initiation of translation.

Supercoiling of DNA involves many steps, but the formation of nucleosomes is an impotant part of this. DNA is wrapped two times around a group of histone proteins.
Also: Transcription is partly regulated by Nucleosomes in eukaryotes.

To which part of an RNA primer can DNA polymerase III enzymes add nucleotides?

DNA polymerase enzymes can only add nucleotides to the 3&rsquo end of a primer.

This diagram shows a simplified diagram of DNA replication.

Which statement best describes what is shown by the diagram?

Replication is in a 5' to 3' direction on the leading strand but the opposite on the lagging strand.

Replication is faster on the lagging strand that the leading strand.

Replication is continuous on the leading strand and disconinuous on the lagging strand.

Replication happens in the same way on both leading and lagging strands.

Continuous DNA replication occurs on the leading strand and discontinuous on the lagging strand.

Which of the following statements give a logical order in the action of just three of the enzymes of the complex group of enzymes which carry out DNA replication?

DNA polymerase III, DNA gyrase, DNA polymerase I

Helicase, DNA polymerase I, DNA polymerase III

Helicase, DNA primase, DNA polymerase III

DNA polymerase I, DNA Gyrase, DNA primase

A complex group of enzymes do DNA replication including helicase, DNA gyrase, single strand binding proteins, DNA primase and DNA polymerases I and III. (also DNA ligase)

DNA gyrase and helicase uncoil and unzip the DNA strand, so that DNA primase can attach a primer.
Then DNA poymerase III adds DNA nucleotides to the 3' end of the primer. The RNA nucleotides of the primer are replaced by DNA nucleotides by DNA polymerase I.

Which is the best description of 'crossing-over'

The exchange of pieces of DNA between sister chromatids of homologous chromosomes

The exchange of pieces of DNA between sister chromatids of autosomes

The exchange of pieces of DNA between non-sister chromatids of homologous chromosomes.

The exchange of pieces of DNA between non-sister chromatids of sex chromosomes.

DNA replication makes a second chromatid in each chromosome in interphase before meiosis.

Crossing over exchanges pieces of DNA between non-sister homologous chromatids and forms new combinations of alleles on the chromosomes formed in meiosis.

The diagram shows two bacteriophage virus particles used in a famous experiment by Hershey and Chase.

Why did they use labelled DNA in one and labelled protein in another?

They were looking for evidence of crossing over in DNA.

They wanted to identify the enzymes and nucleotides in DNA replication.

They wanted to find out whether protein or DNA was inherited by offspring.

They wanted to see which molecule entered the host bacteria's cells.

The Hershey and Chase experiment provided evidence that DNA is the genetic material.

In their experiments they showed that the radioactive label was passed to the DNA in the next generation of bacteriophages.


CORRECT ANSWERS

Cesium chloride gradient centrifugation fractionates macromolecules according to their buoyant densities: at the end of centrifugation, macromolecules of higher and lower densities are closer or farther away from the bottom of the tube, respectively (MCQ 2: A). Density labeling of nitrogen containing macromolecules (MCQ 1: E) with heavy ( 15 N) and light ( 14 N) nitrogen isotopes as described in the protocol of this test generated DNA molecules of three distinct densities: heavy (Peak I), intermediate (Peak II), and light (Peak III). Analysis of the appearance and amount (MCQ 3: C) of these DNA species provided information to describe the mechanism of replication in E. coli cells.

DNA replication, theoretically, could proceed according to three mechanisms [ 3 ]. According to the conservative model (Fig. 3 a), at the end of the replication, the template would remain intact and the daughter DNA would consist of two newly synthesized strands.

Possible models of DNA replication: conservative (a), semiconservative (b), and dispersive (c).

This mechanism would generate heavy and light DNA molecules only. In the semiconservative mechanism (Fig. 3 b), the two strands of the parental DNA molecule would unwind and serve as templates for the synthesis of new, complementary strands. The dispersive model (Fig. 3 c) predicts that both strands of the daughter DNA molecule contain old and newly synthesized regions. In both latter cases, the appearance of DNA molecules of intermediate density is expected after the first replication on the 14 NH4Cl containing medium (MCQ 4: C). Upon heat-denaturation and alkaline cesium chloride gradient centrifugation of Peak II DNA (that triggers and maintains the separation of the DNA strands, respectively MCQ 5: A, MCQ 6: B), two absorption peaks appeared corresponding to heavy and light DNA molecules (MCQ 9: A, MCQ 10: A). This observation strongly supports the semiconservative mechanism (MCQ 11: E). (In the case of dispersive DNA replication, denaturation of Peak II DNA would not change the behavior of the DNA molecules during cesium chloride gradient centrifugation.) The semiconservative model would predict that the ratio of light versus intermediate density DNA would increase in successive DNA replication cycles (Fig. 4 MCQ 12: D). DNA replication was completed in 48 min in this experiment (note the complete disappearance of the original, “heavy” template DNA in Fig. 1 b MCQ 7: A). By 92 min (Fig. 1 c), the second replication cycle was finished (MCQ 8: B).

Density distribution of DNA molecules generated during the first 3 replication cycles of the Meselson-Stahl experiment.