A plasmid Editor

1. Introduction to in silico DNA digestion in ApE [ A plasmid
Editor] and Restriction mapping of DNA
Molecular Biology Lab 5

2. Goals of the Class
• students will learn how to access NCBI [GenBank] database and download DNA
in FASTA file format.
• students will learn how to digest DNA in silico.
• students will learn how to analyse and interpret the results from such digestion
procedures.

3. Open Source Database Definition
• An open source database are available to everyone freely.
• This is the opposite of a proprietary or closed source database in which it is not available for
everyone.
• Some types of open source databases:
• Government and global data
• Financial and economic data
• Crime and drug data
• Health and scientific data
• Academic data
• Business directory data
• Media and journalism data
• Marketing and social media data
• Miscellaneous data

4. Health and scientific databases:
• World Health Organization (WHO)
• Food and Drug Administration
• HealthData.gov
• Broad Institute
• National Cancer Institute
• Centre for Disease Control
• PubMed
• NCBI
• Gene Bank
• Google scholar

5. NCBI
• The National Center for Biotechnology Information advances science and health
by providing access to biomedical and genomic information.
• NCBI has a multi-disciplinary research group concentrating on basic and applied
research in computational molecular biology.
• Together they are studying fundamental biomedical problems at the molecular
level using mathematical and computational methods.
• These problems include gene organization, sequence analysis, and structure
prediction.

6. GenBank
• GenBank ® is the NIH genetic sequence database, a collection of all
publicly available data on the DNA sequences.
• GenBank is part of the International Nucleotide Sequence Database
Collaboration, which comprises the DNA Data Bank of Japan (DDBJ),
the European Nucleotide Archive (ENA), and GenBank at NCBI.
• These three organizations exchange data on a daily basis.

7. Other databases provided by NCBI
• In addition to GenBank, NCBI supports and distributes a variety of
databases for the medical and scientific communities.
• These include the Online Mendelian Inheritance in Man (OMIM), the
Molecular Modeling Database (MMDB) of 3D protein structures, a
Gene Map of the Human Genome, the Taxonomy Browser, and the
Cancer Genome Anatomy Project (CGAP), in collaboration with the
National Cancer Institute, Open Reading Frame Finder (ORF Finder),
Electronic PCR, and the sequence submission tools.

8. DNA Recombination technology
• Technology of the transfer of genetic information(DNA) from one organism to another.
• Basic principles of rDNA technology:
Generation of DNA fragments & selection of the desired piece of DNA.
Insertion of the selected DNA into a cloning vector to create a rDNA or chimeric DNA.
Introduction of the recombinant vectors into host cells.
Multiplication & selection of clones containing the recombinant molecules.
Expression of the gene to produce the desired product.

9. Enzymes used in Recombinant DNA
• Restriction endonucleases are used as molecular scissors, DNA ligase functions to
bond pieces of DNA together, and A variety of additional enzymes.
• Nucleases
• Nuclease enzymes degrade nucleic acids by breaking the phosphodiester
bond that holds the nucleotides together.
• Restriction enzymes are good examples of endonucleases, which cut within a
DNA strand.
• A second group of nucleases, which degrade DNA from the termini of the
molecule, are known as exonucleases.

10. A restriction enzyme
• A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single
stranded DNA at specific recognition nucleotide sequences known as restriction sites.
• They break the phosphodiester bonds that link adjacent nucleotides in DNA molecules.
• HOW RESTRICTION ENZYMES WORKS?
Restriction enzymes recognize a specific sequence of nucleotides, and produce a double-stranded cut in
the DNA, these cuts are of two types:
• BLUNT ENDS.
• STICKY ENDS
• These blunt ended fragments can be joined to any other DNA fragment with blunt ends.

11. NOMENCLATURE OF RESTRICTION ENZYME
• Each enzyme is named after the bacterium from which it was isolated
using a naming system based on bacterial genus, species and strain:
For e.g EcoRI
E = genus Escherichia
co = species coli
R = strain RY13
I= first endonuclease isolated

12. Optimum conditions for activity of Restriction Enzymes
• Optimum conditions are necessary for the expected result:
• Under extreme conditions such as elevated pH or low ionic strength, Restriction
enzymes are capable of cleaving sequences which are similar but not identical to
their recognition sequence.
• Enzyme cut in non specific position

13. Vectors
• Vectors are the DNA molecules, which can carry a foreign DNA fragment to
be cloned.
• These are self replicating in an appropriate host cell.
• Most important vectors are plasmids, bacteriophages, cosmids & artificial
chromosome vectors.

14. Plasmids
• Plasmids are extrachromosomal, double stranded, circular, self-
replicating DNA molecules. Plasmids carry:
1) origin of replication, 2) antibiotic resistance gene(s)
• Usually plasmids contribute to about 0.5%-5.0% of the total DNA of
bacteria.
• A few bacteria contain linear plasmids: Streptomyces sp,
Boreliaburgdorferi. pBR322, pUC
• The plasmids carries genes resistance for ampicillin & tetracycline that
serve as markers for the identification of clones carrying plasmids.

15. ApE (A plasmid Editor) software
• This free software tool allows a user to perform various tasks
on plasmid sequence.

18. How to download your plasmid sequence from NCBI
• Accessing DNA files from NCBI [GenBank]
 Google NCBI GenBank
 GenBank: M77789.2
 This is for pUC19 DNA: 2686 bp
 Send to >> file >>> FORMAT: FASTA >>>> Create File
 FASTA format is a text-based format for representing either nucleotide
sequences or peptide sequences, in which base pairs or amino acids are
represented using single-letter codes.

19. Try digesting the DNA with the following set of enzymes:

20. Go to the enzyme selector:

21. Map your Plasmid:
Digestion of pUC19 plasmid with XbaI and
FspI enzymes.