1 DNA Technology

2 DNA Extraction Chemical treatmentsChemical treatments cause cells and nuclei to burst stickyThe DNA is inherently sticky, and can be pulled out of the mixture “spooling”This is called “spooling” DNA

3 “Spooled” DNA

4 Cutting DNA Restriction enzymesRestriction enzymes cut DNA at specific sequences manageable fragmentsUseful to divide DNA into manageable fragments

5 These cuts usually occur in the following forms:These cuts usually occur in the following forms: 1.The cut can be made straight across a base-pair sequence resulting in a "Blunt End“

6 2. The cut can be made in an offset manner leaving exposed nucleotide sequences. These exposed sequences are called "Sticky Ends"

7 Gene Splicing The presence of sticky ends allows segments of DNA to be joined together.The presence of sticky ends allows segments of DNA to be joined together. Recombinant DNA – joining of DNA from 2 or more speciesRecombinant DNA – joining of DNA from 2 or more species DNA strands which have been cut by the same restriction enzyme can easily bond together according to base pairing rules.DNA strands which have been cut by the same restriction enzyme can easily bond together according to base pairing rules.

8 Gene splicing cont’

9 Gene splicing use: Allows genes to be "cut & pasted" between organisms. –E.g. production of human insulin. The DNA sequence of insulin is identified, & cut out with restriction enzyme. A plasmid from E. coli is removed & cut open using the same restriction enzyme Both fragments have complimentary sticky ends, they bind, the gene for human insulin is integrated into plasmid The plasmid is reinserted into bacterial cell which produces insulin. Human insulin can now be extracted and provided to diabetics.

Plasmid – a ring of DNA found in a bacterium in addition to its main strand of DNA 10

11 Electrophoresis size & chargeDNA can be separated based on size & charge phosphate groups negativelyThe phosphate groups are negatively charged gel electricityDNA is placed in a gel and electricity is run through

12 Electrophoresis machine

13 Electrophoresis Negative DNANegative DNA moves toward the positive end Smaller farther and faster through poresSmaller fragments move farther and faster through pores

14 Electrophoresis

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Tutorial with animation cience/sbioac/genetics/Electro.ht mhttp:// cience/sbioac/genetics/Electro.ht m 16

17 Animations namaker/animations/Chrom%26 Elpho.htmlhttp:// namaker/animations/Chrom%26 Elpho.html

18 Steps in DNA Sequencing Many copies of a single strand of DNA are placed in a test tubeMany copies of a single strand of DNA are placed in a test tube DNA polymerase is addedDNA polymerase is added A mixture of nucleotides is added some of which have dye molecules attachedA mixture of nucleotides is added some of which have dye molecules attached Each base (A,T,C,G) has a different color dyeEach base (A,T,C,G) has a different color dye

19 Steps in DNA Sequencing some dyed nucleotidessome regular onesBy chance, some dyed nucleotides & some regular ones are added stop the chainDye molecules are large and stop the chain from growing

20 DNA Sequencing multiple sizes with colors that can be identifiedThe result is DNA fragments of multiple sizes with colors that can be identified

21 DNA Sequencing After the gel separates the resulting fragments by size, we 'read' the sequence from bottom to top.After the gel separates the resulting fragments by size, we 'read' the sequence from bottom to top.

22 DNA Fingerprinting DNA is now a powerful tool in identification.DNA is now a powerful tool in identification. Based on the fact that the amount of "junk DNA" differs uniquely between individuals.Based on the fact that the amount of "junk DNA" differs uniquely between individuals. Structural genes are often separated by large regions of repeating base pairs.Structural genes are often separated by large regions of repeating base pairs. The number of these repeats is unique to an individual.The number of these repeats is unique to an individual. When DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to that individual.When DNA from a person is cut with a restriction enzyme, the length of the fragments will be unique to that individual.

23 Fingerprinting cont’ This will produce a unique banding pattern following a gel electrophoresis.This will produce a unique banding pattern following a gel electrophoresis. This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000.This test is highly accurate, and the probability of another individual possessing an identical banding pattern is estimated as around 1:14,000,000,000.

24 Fingerprinting cont’

25 Copying DNA Polymerase Chain ReactionPolymerase Chain Reaction Also called PCR A method of making many copies of a piece of DNA

26 Steps in Copying DNA A DNA molecule is placed in a small test tube DNA polymeraseDNA polymerase that can work at high temps is added

27 Steps in Copying DNA DNA is heatedThe DNA is heated to separate the two strands PrimersPrimers, short pieces of DNA complementary to the ends of the molecule to be copied, are added

28 Copying DNA DNA polymerase adds new bases to the separated strandsThe tube is cooled, and DNA polymerase adds new bases to the separated strands

29 PCR Large amounts of DNA can be made from a small starting sample

PCR Virtual Lab tent/labs/pcr/ tent/labs/pcr/ 30

31 Cloning CloneClone- a member of a group of genetically identical cells asexual reproductionProduced by asexual reproduction (mitosis)

32 Cloning organisms body cell egg cellA body cell from one organism and an egg cell from another are fused divides like a normal embryoThe resulting cell divides like a normal embryo (many identical cells produced)

33 Cloning “Dolly”

GEL ELECTROPHORESIS VIRTUAL LAB tent/labs/gel/ tent/labs/gel/ 34

35 REFERENCES -