Chapter 11 Molecular tools and techniques

1. Chapter 11Molecular tools and techniques DNA Profiling Restriction enzymes Gel electrophoresis The Southern Blot technique DNA amplification- Polymerase Chain Reaction (PCR) Sequencing DNA.

2. Gene technology refers to the manipulation and use of DNA. • DNA profiling is characterising the genetic makeup of an individual by cutting DNA into fragments using restriction enzymes and examining the banding pattern on an electrophoresis gel. • Enzymes are important in all gene technologies. (What a surprise!!) • The enzymes used have been discovered in, and extracted from the cells of • eukaryotes, • prokaryotes • and viruses.

3. Restriction enzymes • Restriction enzymesare made by bacteria to protect themselves from viruses. They inactivate any viral DNA by cutting it in specific places. • If foreign DNA enters the bacteria cell the restriction enzyme will cut it up into small pieces. A process known as restriction. They cut up only certain base pair sequences and thus are handy in genetics. • Restriction enzymes can be used to cut DNA at specific sequences called recognition sites. They then rejoin the cut strands with DNA ligase to form new combinations of genes.

4. Recognition Site cut Recognition Site The restriction enzyme EcoRI cuts here cut cut Restriction Enzymes • Restrictionenzymes are one of the essential tools of genetic engineering. Purified forms of these naturally occurring bacterialenzymes are used as “molecularscalpels”, allowing genetic engineers to cut up DNA in a controlled way. Also called endonucleases. • Restrictionenzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites (see below). • By using a ‘tool kit’ of over 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers are able to isolate and sequence DNA, and manipulate individual genes derived from any type of organism. GAATTC GAATTC DNA CTTAAG CTTAAG

5. Specific Recognition Sites • Restriction enzymes are named according to the bacterial species they were first isolated from, followed by a number to distinguish different enzymes isolated from the same organism. • e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H.

6. A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site:

7. Cutting specificity • When DNA is cut with a restriction enzyme the resulting fragments are either left with a short overhang of single stranded DNA called a ‘sticky end’ or no overhanging DNA called a ‘blunt end’. GAATTC GTTAAC CTTAAG CAATTG EcoRI – leaves a sticky end. HpaI – leaves blunt ends

8. A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site A A T T C G C T T A A G The cuts produce a DNA fragment with two “sticky” ends A A T T C A A T T C G G The two different fragments cut by the same restriction enzyme have identical sticky ends and are able to join together G G C T T A A C T T A A When two fragments of DNA cut by the same restriction enzyme come together, they can join by base-pairing Sticky Ends • It is possible to use restriction enzymes that cut leaving an overhang; a so-called “sticky end”. • DNA cut in such a way produces ends which may only be joined to othersticky ends with a complementary base sequence. • See steps 1-3 opposite: Restriction enzyme: EcoRI Fragment A A T T C G C T T A A G Restriction enzyme: EcoRI Sticky end DNA from another source

9. C C C C C C G G G G G G G G G C C C G G G G G G C C C C C C DNA G G G C C C C C C G G G G G G C C C C C C G G G G G G C C C cut cut C C C G G G C C C G G G Blunt Ends Recognition Site Recognition Site • It is possible to use restrictionenzymes that cut leaving no overhang; a so-called “blunt end”. • DNA cut in such a way is able to be joined to any other blunt end fragment, but tends to be non-specific because there are no sticky ends as recognition sites. Restriction enzyme cuts here The cut by this type of restriction enzyme leaves no overhang DNA from another source A special group of enzymes can join the pieces together

10. Two pieces of DNA are cut using the same restriction enzyme. The two different DNA fragments are attracted to each other by weak hydrogen bonds. This other end of the foreign DNA is attracted to the remaining sticky end of the plasmid. A A T T C G Plasmid DNA fragment Foreign DNA fragment G C T T A A Ligation • DNA fragments produced using restriction enzymes may be reassembled by a process called ligation. • Pieces of DNA are joined together using an enzyme called DNA ligase. • DNA of different origins produced in this way is called recombinant DNA because it is DNA that has been recombined from different sources. • Steps 1-3 are involved in creating a recombinant DNA plasmid:

11. Gel Electrophoresis • Is one of the most commonly used tools. • It is a molecular separating technique used to sort molecules – DNA and proteins on the basis of size, electric charge, and other physical properties. Brief outline: • DNA is extracted from cells. • It is cut using restriction enzymes. • Added to a gel tank. • Electric current is run through it. • DNA fragments migrate towards the positive electrode. • Smaller fragments move faster.

12. DNA samples are loaded into wells

13. DNA is stained using ethidium bromide or methylene blue. Electrical current applied to the chamber

16. Polymerase chain reaction To work with DNA it is necessary to have more than a few molecules. The polymerase chain reaction (PCR) is a technique used to amplify (make millions of pure copies of) a piece of DNA in a test tube (Figure 11.4). It allows, for example, forensic scientists to amplify the DNA in traces of blood left at the scene of a crime. It is also used to amplify a particular gene from a sample of DNA fragments. Biologists developed the technique of PCR by studying how DNA is synthesised naturally in cells.

17. The technique of (PCR) • PCR is a chain reaction of DNA replication events. The method uses a complex mixture of ingredients, which is heated and cooled in cycles. At each cycle of synthesis, the number of copies of the DNA fragment is doubled. A large amount of DNA can be produced in a test tube in a few hours. • The polymerase chain reaction (PCR) mixture contains four ingredients: • a sample of DNA, which acts as a template to make millions of copies • a source of the four nucleotides: A, T, C and G, which are the building • blocks for DNA replication • a DNA polymerase (Taq polymerase), which is a heat-resistant enzyme • single-stranded DNA primers, which are synthetic, short pieces of DNA • that are complementary to sequences of bases that flank the DNA region to be amplified. Primers specify the starting and finishing points for DNA replication (see Chapter 10).

18. These ingredients are placed together in a plastic tube in a DNA thermocycler, a heating block that is able to change temperature very rapidly. The thermocycler initially heats to a temperature of 95°C. This breaks the hydrogen bonds and separates the strands of the DNA sample to make two single-stranded templates (Figure 11.5). The thermocycler then cools to a temperature of 50–60°C to allow the primers to bind to their complementary DNA sequence. These primers are typically 18–30 nucleotides in length. The two primers bind at the ends of the DNA that is to be amplified; one primer binds to each template strand.

19. The temperature is then increased to approximately 72°C, the optimal temperature for the DNA (Taq) polymerase enzyme. The enzyme DNA polymerase begins to move along the template DNA, starting from the primer and adding nucleotides. Nucleotides are added at the 3′ end of the new strand according to the complementary base-pairing rules. Once this first round of DNA replication is complete, two double-stranded DNA molecules have been produced from each double-stranded DNA molecule added to the reaction mixture. The thermocycler then heats to 95°C and the next cycle of strand separation, binding of primers and DNA replication begins. Thermocyclers can be programmed to go through many cycles. Typically, 30–40 cycles are used to amplify a DNA sample.

22. DNA samples can be analysed to explore possible paternal and maternal relationships between parents and children. This is possible because half of each person's 23 pairs of chromosomes come from their mother, and half from their father. • Each of the chromosomes contains many sections of non-coding DNA that does not seem to code for a protein, but contains areas called short tandem repeats (STRs). Each STR contains repeats of short sequences of bases, such as CATG in CATGCATGCATG. • When STRs are tested in DNA profiling, they occur in pairs. One chromosome in a pair carries an STR from the person's mother, and the other carries an STR from the person’s father. • http://www.biotechnologyonline.gov.au/popups/int_dnaprofiling.html

23. A person’s DNA profile as seen on an electrophoresis gel usually shows two lines for each of the STRs tested. This is because usually, the STRs inherited from the parents are of different lengths. Occasionally, only one line appears because both STRs in a pair are of the same length. • When the DNA profile of a child is compared to the profiles of the child's genetic parents, it is possible to match one line in each STR area with a line in that area of the mother's profile. In this way, DNA profiling can also reveal non-paternity. • Using 10 different STR loci allows for a person to be identified with a very high level of probability. In America they use 13 STR loci due to their larger population to increase accuracy.

24. DNA sequencing • DNA sequencing is used to work out the exact order, or sequence, of the base pairs in a section of DNA. • Knowing the base sequence can be helpful if you want to locate a specific gene by using a gene probe, or to make an artificial chromosome with a specific gene on it. • DNA sequencing is also being used to identify and locate all the genes in an organism. (Eg: Human Genome Project) • A DNA sequencing machine uses the same principle as electrophoresis. However, it is so sensitive that it can separate DNA strands that differ in length by only one nucleotide.

25. The base sequence of a strand of DNA is worked out by: • copying the DNA many times, each time constructing DNA chains of different lengths • using electrophoresis to separate the strands from shortest to longest.

26. Recombinant DNA technology • Often called genetic engineering. • It involves the removal of DNA from one organism to be placed in another organism of the same or a different species. • The organism that expresses the foreign genes is called a transgenic organism. • This technology requires: 1. a way of extracting or producing the desired gene. 2. a way of making many copies of the DNA. 3. a way of inserting the desired gene into the target organism.

27. Recombinant DNA Technology • The major tools of recombinant DNA technology are bacterial enzymes called restriction enzymes, which were first discovered in the late 1960s. • These enzymes protect bacteria against intruding DNA from other organisms. • They work by cutting up the foreign DNA, a process called restriction. • If foreign DNA enters the bacteria cell the restriction enzyme will cut it up into small pieces. They cut up only certain base pair sequences and thus are handy in genetics • The bacterial cell protects its own DNA from restriction by adding methyl groups (--CH3) to adenines or cytosines within the sequences that would otherwise be recognized by the restriction enzyme.

28. PLASMIDS Plasmids are molecules of DNA that are found in bacteria, separate from the bacterial chromosome. They: • are small (a few thousand base pairs) • usually carry only one or a few genes • are circular • have a single origin of replication

29. Sex pilus conducts the plasmid to the recipient bacterium A plasmid about to pass one strand of the DNA into the sex pilus Plasmid of the non-conjugative type Plasmid of the conjugative type Plasmid DNA (a reminder) Recipientbacterium • Bacteria have small accessory chromosomes called plasmids. • Plasmids replicate independently of the main chromosome. Donorbacterium

30. Plasmids are able to transfer from cell to cell. E coli plasmid is used to replicate the insulin gene. This is then placed in a bacteria cell and cultured and insulin removed. By using the same restriction enzymes on humans and bacteria the same DNA is cut this means that the gap from the bacteria DNA can be filled by the human insulin gene. Insulin

31. The insulin gene can be isolated using restriction enzyme as scissors to cut the insulin gene from the rest of the human body. We then manufacture insulin. To do this we take the gene cut by the restriction enzyme and put in a plasmid which are also from bacterial cells. Restriction Enzyme used in insulin

32. Human gene Sticky end Sticky end Restriction enzyme recognition sequence A gene of interest (DNA fragment) is isolated from human tissue cells Ampicillin-resistance gene Tetracycline-resistance gene Human DNA and plasmid are treated with the same restriction enzyme to produce identical sticky ends Gene disrupted An appropriate plasmid vector is isolated from a bacterial cell Sticky ends Restriction enzyme cuts the plasmid DNA at its single recognition sequence, disrupting the tetracycline resistance gene Mix the DNAs together and add the enzyme DNA ligase to bond the sticky ends Recombinant DNA molecule Human gene Recombinant plasmid is introduced into a bacterial cell by simply adding the DNA to a bacterial culture where some bacteria take up the plasmid from solution Escherichia coli bacterial cell Using Plasmids Preparation of the Clone Human cell Plasmid Chromosome DNA in chromosome Plasmid vector

33. Applications of DNA profiling. • Establishing parentage. • Establish other family relationships. • Analyse biological evidence from a crime scene. • Establish the level of genetic variation in threatened species. • Establish evolutionary relationships between groups of organisms.

34. Application of DNA profiling • The use of DNA techniques in forensic science has advantages over other techniques. This is because DNA is a stable molecule, which can withstand drying out. • The PCR technique allows forensic scientists to work with very small amounts of DNA

35. Application of DNA profiling • DNA profiling is used to reveal family relationships in paternity tests or to test members of a family for a possible inherited disorder. • Estimating the amount of genetic variation in rare and endangered species is useful for determining a conservation management strategy. • Detailed information is provided by gene sequencing, which has many applications such as in crop breeding and medicine.

36. DNA profiling This is also called DNA fingerprinting. It uses a pattern of repeated DNA sequences (called ‘short tandem repeats’ – STR’s) that are unique to an individual to identify a particular person’s DNA. They are between genes and are in the non-coding DNA. There are more than 10,000 STR loci in one set of human chromosomes. The DNA profile is observed using gel electrophoresis technology.