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Cell Biology

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A set of questions based on the restriction enzyme and PCR practicals:

1) Restriction enzymes

In 1968, Dr. Werner Arber at the University of Basel, Switzerland and Dr. Hamilton

Smith at the JohnsHopkinsUniversity, Baltimore, discovered a group of enzymes in

bacteria, which when added to any DNA will result in the breakage [hydrolysis] of the

sugar-phosphate bond between certain specific nucleotide bases [recognition sites].

This causes the double strand of DNA to break along the recognition site and the DNA molecule becomes fractured into two pieces. These molecular scissors or “cutting” enzymes are restriction endonucleases.

Two common restriction enzymes (endonucleases) are EcoRI and PstI which you will use in the practical sessions. The diagram below illustrates how a restriction enzyme works:

The line through the base pairs represents the sites where bonds will break if the restriction endonuclease EcoRI recognizes the site GAATTC. The following analysis questions refer to how a piece of DNA would be affected if a restriction endonuclease were to "cut" the DNA molecule in the manner shown above.

1. How many pieces of DNA would result from this cut?

Two double-stranded DNA fragments would be produced.

2. Write the base sequence of the DNA fragments on both the left and right side of the “cut”.

Since we are creating fragments with 5' overhangs ("sticky ends"), it is probably best to write this down as double-stranded DNA. The convention is to represent DNA 5' to 3' (for the top line). An evenly-spaced font like Courier is best for good alignment.

5' ATG

TACTTAA 

and 

5' AGGTAATTGAG

TCCATTAACTCTTAA

Note that the description above lacks information on the phosphorylation status of the termini. This may be relevant for subsequent enzymatic steps (labeling, ligation etc).

3. Consider the two samples of DNA shown below - single strands are shown for simplicity:

Sample #1

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

Sample #2

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

If both samples are treated with the restriction enzyme EcoRI [recognition sequence

GAATTC] then

a) indicate the number of fragments and the size of each fragment from each sample of DNA.

These DNA molecules are each cut into two pieces.

Sample 1:   fragments of 11 bp and 13 bp   (plus some overhang).

Sample 2:  fragments of 5 bp and 19 bp   (plus some overhang).

b) List fragment size in order: largest ——> smallest

19, 11 and 5 bp

2. Gel electrophoresis

1. In the diagram above, what is contained in each band ?

Assuming this is an agarose gel stained with EthidiumBromide or SYBR Green then we are looking at nucleic acid, which could be DNA or RNA. Each band represents nucleic acid with specific molecular weight (for which size, in basepairs, is a proxy).

Of course, the drawing could also present a Northern, Southern, or Western Blot, or an autoradiogram of radiolabelled DNA…

Remarkably, a lane with size markers appears to be missing, but then, again, this may be a routine gel   also; adding xylene cyanol and bromephenol blue to the sample buffer prevents material to run off the gel.

2. Which of the DNA samples have the same number of restriction sites for the restriction endonucleases used? Write the lane numbers.

Lanes 2, 3 and 4 all show patterns resulting from a fragment with one restriction site.

3. Which sample has the smallest DNA fragment?

Lane 5.

4. Assuming a circular piece of DNA (plasmid) was used as starting material, how many restriction sites were there in lane three?

It must have had two sites.

5. Which DNA samples appear to have been “cut” into the same number and size of fragments?

CS and S1.

6. Based on your analysis of the gel what is your conclusion about the DNA samples in the drawing? Do any of the samples seem to be from the same source? If so, which ones? Describe the evidence that supports your conclusion.

It is conceivable that all lanes show digests from the same plasmid, but it is difficult to be affirmative without size markers.  Sample 5 is problematic - the sum of the two lowest bands must add up to the lower band in lane 3.

I assume that we are not looking at doublets. Since a complete digest produces equimolar fragments, fluorescent intensity is simply dictated by size (i. e. decreasing from top- to-bottom bands). Because of this, it is normally easy to spot "odd bands".

3. Restriction enzymes practical

Refer to Table 1, which gives the distances travelled by fragments created by the restriction enzymes.

  1. Create a standard curve using the distance (x-axis) and

fragment size (y-axis) data from the HindIII lambda digest (DNA marker).

Using the semilog graph paper provided, plot distance versus size for bands 2–6 produced by the size marker DNA. Draw a line of best fit through the points. Extend the line all the way to the righthand edge of the graph.

Two comments:  (1) Not sure that extrapolation to the right is reliable for this type of gel. (2) Not sure either that professionals still use semi-log paper. Probably it is easier to enter the data in two Excel columns, log-convert the bps and do a linear regression to check the regression coefficient prior to intrapolations.

  1. Why does lane 2 have only a single band?

This is uncut, linear phage DNA. Note that uncut, circular plasmid DNA often does not run as a single band.

  1. Why do the H lane and the E lane have different numbers of bands?

Because the phage lambda genome has a different set of restriction sites (number, locations) for these two enzymes, which have different recognition sites.

  1. Using your standard curve, estimate the sizes of one band from each of the enzyme lanes – so three examples in total. 

Table 1 

 

 

 

L = uncut lambda

P = PstI enzyme

E = EcoRI enzyme

H = HindIII enzyme

 

Distance (mm)

Base pairs

Distance (mm)

Base pairs

Distance (mm)

Base pairs

Distance (mm)

Base pairs

Distance (mm)

Base pairs

Band 1

10

23,130

9

 

11

 

10

 

10

 

Band 2

11

9,416

 

 

15

 

13

 

11

9.4x103 bp

Band 3

13

6,557

 

 

18.5

 

14

6.4x103 bp

13

 

Band 4

15

4,361

 

 

19

2.0x103 bp

14.5

 

15

 

Band 5

20

2,322

 

 

21

 

16

 

20

 

Band 6

21

2,027

 

 

22

 

 

 

21

 

PCR practical 

  1. Draw a labelled gel diagram showing the results of this practical.
  2. What do the bands in the allele ladder represent ?
  3. Why do the lanes other than the allele ladder lane contain two bands ?
  4. Do any of the samples from the suspects match the crime scene sample?

Exam

The structure of the DNA molecule. How do the bases pair up?

DNA is a double helix consisting of a desoxyribose-phosphate backbone and four different bases that pair between the strands. Maxim-Gilbert base-pairing says G (guanine) pairs to C (cytosine), and A (adenine) pairs to T (thymidine). These possibilities exist because of the potential to form matched hydrogen bridges; also, the number of aromatic rings between the DNA backbones is then always the same (three).

Which bases are purines and which are pyrimidines?

A and G are purines (single aromatic ring), C and T are pyrimidines (two rings).

The structural differences between DNA and RNA.

Genomic DNA is normally double-stranded (except in some phages), RNA is single-stranded, again, with the exception of some viruses such as Influenza, which have a dsRNA genome. Single-stranded RNA engages in considerable intramolecular folding, its so-called secondary structure. DNA is assembled from desoxyribonucleotides, RNA from ribonucleotides. RNA has no C (cytosine) but U (uracil) bases instead, which pairs with A. DNA is used to store genetic information in most organisms and stored as such in the nucleus and (shorter sequences) in mitochondria and chloroplasts. RNA is fragile in the sense that it is rapidly degraded by ubiquitous, robust enzymes called RNAses, and mRNAs (messenger RNAs) in the cell are typically short-lived. Another important class of RNAs are ribosomal RNAs; ribosomes are structures that "translate" mRNA into proteins.

The basic structure of the chromosome.

DNA is wrapped up by histones, evolutionary well-conserved, basic (positively charged) proteins. These are then further organized in nucleosomes. Overall, DNA is very tightly packed in the nucleus. Stretched out, a single cell's DNA would cover a meter or so.

How DNA is copied, and how errors are dealt with.

DNA replication is semi-conservative, i. e. each strand is copied by adding complementary nucleotides. DNA replication is very precise, with multiple "proof-reading" enzymes checking the process (more precise than, for example, transcription). However, it is sensitive to ionizing irradiation or free radicals. Damage to bases can sometimes be dealt with by enzymes that scan for chemical modifications (e. g. UV-induced T-T adducts), then correct the error by "looking" at the complementary base.

Restriction enzymes and what they do.

Restriction enzymes are made by micro-organisms. The enzymes probably evolved to protect the host from viral infection (bacteriophages) or other unfriendly invasions of DNA. Bacteria protect their own genome from degradation by methylating the same restriction sites. Each enzyme has a well-defined recognition site, but there are many of them, and there is quite a bit of variability with 4, 6 or 8 bp cutters. There are redundancies (e. g. GANTC, where N can be any nucleotide). Some enzymes leave 5' overhangs (like EcoRI), some 3' overhangs (PstI) or produce blunt ends (SmaI). These enzymes have been extremely valuable for molecular cloning of DNA, especially before PCR was devoloped.

The difference between complementary and genomic DNA libraries.

Complementary DNA (cDNA) is made by "reverse-transcribing" RNA (usually mRNA), typically with the help of an avian retroviral enzyme and an oligo-dT (or a random) primer. Next, the RNA is degraded (RNAse), a complementary DNA strand is synthesized, adapters are ligated and cut with restriction enzymes for cloning in a plasmid with a selectable marker for transfection into bacteria. A genomic DNA library represents DNA fragments from (usually) nuclear DNA, cloned in much the same way as above. So, cDNA libraries represent an organisms "transcriptome" (for a given cell/tissue); obviously, there is a risk that some transcripts (e. g. for actin) are overrepresented, whereas genomic libraries represent an organisms nuclear DNA- the latter is the same in all somatic cells.

DNA sequencing.

The oldest method ("Maxam & Gilbert") used chemical approaches on end-labelled, radioactive DNA fragments. Newer methods are all based on the Sanger approach, wherein DNA is replicated in (four) reactions spiked with di-deoxynucleotides, which halt further transcription. The four "ladders" of transcripts, usually labelled with fluorescent markers, can be run on tall, denaturing, urea-containing, thin polyacrylamide gels for reads of up to a few hundred bps per reactions (on multiple gels). These days, however, the process is highly automated (no gels are used anymore).

The polymerase chain reaction.

This is a chain reaction where an excess of short oligonucleotides that hybridize either ends of a "target DNA" molecule, which may occur in just a few molecules or so, in multiple rounds of "melting" (at 94 C or so) the strands apart, annealing with the primers and transcription. Each cycle (potentially) amplifies the previous amount of DNA by factor two, for 25-40 cycles or so. Initially the process was painstaking, requiring scientists to move tubes between waterbaths and reading enzymes. This all changed with the discovery of heat-stable DNA polymerases and programmable heating blocks.

The translation of DNA sequences to amino acids.

DNA is first transcribed into messenger RNA. The RNA is usually spliced (removal of introns), and a poly-A tail is added, as well as a 5' methyl "cap" (increase stability). The mRNA is then transported out of the nucleus to the cytoplasm where ribosomes will scan for the translation initiation site (usually the first, 5', ATG triplet) and polymerize amino acids as instructed by subsequent triplets. The process ends when stopcodons are found. Proteins can then be processed for export (actually, this is already encoded by the first 20 or so amino acids) or moved to the Golgi for other posttranslational modifications.

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