S12 Nucleic Acid Technology

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LSM1401 Summary 12 © Lim Fang Jeng

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Nucleic Acid Technology

Four classes of enzymes involved in manipulating DNA: (1) Polymerase : Extend/replicate

(2) Nuclease : Degrade/cleave (3)_Ligase : Join

(4) Modifying enzyme : adding/removing chemical groups

Restriction Enzyme Mapping

- Restriction Enzyme (Endonuclease)

o Cleave short recognition sequences (4 to 8 bp) on DNA o Very specific

o Most recognition sites are symmetrical (palindromic) o Generates terminals bearing 5’ phosphate and 3’ OH o requires Mg²⁺^{and ATP}

- Gel electrophoresis

o Uses ions to separate and determine the length of the DNA cleaved by some restriction enzymes

o DNA is negatively charged

o Smaller fragment will tend to go down the tube attracted by the cation

- Read DNA sequence from below to above! - We can investigate which position the restriction

enzyme cleaves the DNA

Southern Blotting

Southern Blotting = RE cuts + Gel Electrophoresis + Labelled Probe Hybridization

= Detection of Specific DNA fragment - Transfer DNA from gel to a nitrocellulose paper

- The DNA is transferred to the paper by capillary action

- After transferring to the paper, a probe can be added to recognize the sequence

- This involves complementary base pairing

- The gene with greater homology will have greater hybridization and hence enable us to see the probe

Restriction-Fragment Length Polymorphism (RFLPs)

- A change of one base pair could eliminate a restriction site - If there is a mutation like such, RLFP can enable us to recognize

the mutation site (after Southern blotting, Electrophoresis, Hybridization)

- We can diagnose the disease.

- Can determine the genuine lineage (determine who is the child’s real parents)

Blunt end Sticky ends

(+) (-)

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Polymerase Chain Reaction

- Useful when we don’t have enough sample

- The small sample DNA serves as a template for DNA polymerase - Make complementary primers (DNA primers – more stable)

- Elongation by DNA polymerase (Taq – enzymes that is stable during high temperature) -_Steps:

(1) Heat DNA to 90^oC to denature it

(2) Primers anneal to template strand (55^o^C-65^o^C)

(3) DNA polymerase extend/synthesize complementary strand (70-72^o^C)

Application to DNA Fingerprinting

- Some region in the DNA of an individual has a short tandem repeat - two or more nucleotides are repeated (typically in non-coding region) - We have different STR, so it can be determined of identification

- PCR can be used to amplified the sample, then can be used in Crime Scene Investigation (visualized by Gel electrophoresis)

DNA Sequencing

Restriction enzyme Construction of DNA library DNA sequencing of individual clones by DNA library - If we have a large chunk of DNA, we use restriction enzyme

- Incorporate into bacterial plasmids (Self-replicating, circular DNA, carry non-essential genes often found in prokaryotes) by ligase

- Specific clones can be selected by colony hybridization - Remaining clones are saved and put in DNA library

Colony Hybridization

- Replicate DNA onto nitrocellulose disc - Transfer to nitrocellulose paper - Add probe, X-ray screening

- Spot the position of the desired gene

cDNA Library

- Uses mRNA as template, intron is absent (due to splicing of mRNA) - Reverse Transcriptase  cDNA

- Incorporate into bacterial plasmids and selection - Remaining clones are saved in cDNA library

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Principle of DNA sequencing

- Base on synthesizing new strand of DNA (complementary to the template)

- Now, dideoxy-ribonucleotide is introduced (THEY ARE THE ONLY ONE LABELLED WITH PROBES) - It will terminate the sequence since it has no 3’-OH

- Enable sequencing possible

- By this, as the base pairing stops, we can detect the ddNTP as the base and recognize it by probe

Recombinant DNA Technology

Cloning

- Introduce foreign DNA into host -_Steps:

(1) Cut DNA sequence with restriction enzyme

(2) Insert into vector such as plasmid (antibiotic resistance gene), which will help to select clones later on (3) Seal sticky ends of plasmid and DNA sequence with ligase

(4) Transformation/Transfection into the host

(5) Selection of host-cells carrying the recombinant DNA by Blue-white bacteria selection (6) Propagate the correct clone

Blue-White Bacteria Selection

- Selection based on the antibiotic resistant gene - Label the desired gene for production as blue

- Foreign DNA with sticky ends is inserted between the blue-labelled gene and mixed with the original plasmids

- After that, insert the bacterial cells to the antibiotic solutions, 3 outcomes will result:

(1) No plasmid observed (sensitive to antibiotic) (2) White-coloured sample (The desired gene is not

produced)

(3) Blue-coloured sample (Desired gene)

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Microarray Technology

- Measure the expression level of certain gene to diseases - Targeted for mRNA

- Placing specific nucleotide sequences - Involves complementary base pairing

- The intensity of the fluorescence is used to quantity the amount of cDNA bound to a specific probe - Poly T primer is used to reverse transcribe the cDNA

- We can track the gene that expresses the disease by seeing the colours