Molecular Biology II

1. Molecular Biology II Common Techniques

2. Aspects to Cover DNA/RNA Quantitation Gel Electrophoresis Restriction Endonuclease Digestion DNA Ligation DNA/RNA Polymerases Reverse Transcription (RT) Plasmid Vectors Cloning Southern/ Northern Blotting Primers Polymerase Chain Reaction (PCR) Quantitative RT-qPCR Quick application of RT-qPCR in our laboratory

3. Restriction Endonuclease Digestion Cut DNA at specific 4 or 6 base pair sequences Sequences are usually palindromic C C C G G G Can create ‘sticky’ or ‘blunt’ ends G G G C C C SmaI C C C G G G C C C G G G

4. Restriction Endonucleases

5. DNA Ligation DNA ligase can join a break in the sugar – phosphate backbone of DNA

6. Polymerases Synthesis of nucleic acids from template in the 5’ to 3’ direction DNA-dependent DNA polymerases (replicate DNA) DNA pol a,b,g,d,e – replication of DNA in eukaryotes (19) DNA pol I, II, III – replication of DNA in prokaryotes Klenow – subunit of E. coli DNA pol I labelling fragments for Southern/Northern blot Taq DNA pol – from Thermus aquaticus PCR DNA-dependent RNA polymerases (transcribes RNA) Reverse Transcriptase Uses RNA as template for DNA synthesis

7. Reverse Transcriptase RNA-dependent DNA polymerase: synthesises DNA from RNA template Major constituent of retroviruses: facilitates insertion of viral genome into host genome 5`-CGAUCGGAUCCAGCUGGACGCUAGCGUAAAAAAAA-3` 5`-CGATCGGATCCAGCTGGACGCTAGCGTAAAAAAAA-3` TTTTTT-5` 3`-GCTAGCCTAGGTCGACCTGCGATCGCATT RT RNAseH activity of Reverse Transcriptase degrades RNA strand DNA strand can act as template for second strand synthesis

8. A G C A U G C U A G C A C G G C U A Reverse Transcription (RT) Reaction requires a number of different components: Appropriate buffer conditions - [salt], MgCl2, dNTPs Primers: non-specific - oligo dT, random hexamer gene-specific - designed for target gene Reverse Transcriptase enzyme: M-MLV, AMV RNaseH RNA must be heated to 65oC to remove secondary structure Used predominantly to generate complementary DNA (cDNA) as first step in RT-qPCR mRNA quantitation or in cloning

9. Plasmid Vectors Small circular molecules of dsDNA Frequently used for cloning due to their ability to carry foreign DNA into bacterial cells and create multiple copies Contain multiple cloning sites to assist in insertion of foreign DNA Contain regulatory elements for replication and antibiotic resistance genes for selection Used as vectors to express cloned genes in both bacterial and eukaryotic cells

10. Cloning When the host cell divides, copies of the vector are passed to the progeny Plate bacterial host on agar and allow time for multiple cell divisions to form a colony (clone). Each cell in the clone contains one or more copies of the vector and gene. The initial fragment is now said to be cloned. A fragment of DNA is ligated into a circular DNA molecule (vector), creating a recombinant DNA molecule. The vector is transformed into a host cell (bacteria) The bacteria replicates the vector The plasmid and the insert can be then isolated in bulk for subsequent procedures – further cloning, sequencing, Southern/Northern blotting etc

11. Southern Blotting Originally named after Edward Southern Hybridise labelled probe (from target gene) to membrane and wash away unbound probe Isolate genomic DNA from organism of interest Digest DNA with restriction enzymes and electrophorese Enables detection of specific DNA sequences amongst an unknown group of sequences – helps assess gene complexity and copy number within a genome Visualise bound probe and determine complexity of gene by size and number of fragments Transfer fractionated DNA to nitrocellulose membrane by capillary transfer in alkaline buffer to denature DNA strands Enables detection of related but not identical sequences by variation in wash stringency Each different genomic digest has two hybridising fragments suggesting two copies of the gene (unless each restriction site occurs in the probe sequence) Each different genomic digest has one hybridising fragment suggesting a single copy of the gene

12. Northern Blotting Early example of scientific humour – virtually identical to Southern blotting but using RNA isolated from cells instead of DNA Determines whether a gene is transcribed, what size the transcript is and to what extent – level of RNA expression Important to remember that is a snapshot of expression levels, is a combination of synthesis and degradation of RNA Isolate RNA and electrophorese Transfer to membrane Hybridise with gene-specific labelled probe Visualise

13. Primers Used in reverse transcription, sequencing, PCR Short pieces of DNA (18-25 bp) used to “prime” for DNA synthesis Provide 3` OH group for strand elongation Must be complementary to region in template Can either target a specific gene (PCR) or randomly prime (RT) If targeting a specific gene, must not be complementary to any other region in template

14. Polymerase Chain Reaction (PCR) Used to clone specific sequences of DNA for further manipulation Used to in conjunction with reverse transcription to quantitate levels of a specific gene mRNA (RT-qPCR)

15. 94oC 94oC 50-65oC 72oC Polymerase Chain Reaction Taq polymerase synthesizes DNA complementary to template in 5` to 3` direction The use of two primers allows targeting of specific sequences Targeted DNA replication using thermostable DNA polymerase Primers are complementary to opposite strands of target region but not complementary to any other sequences DENATURE ANNEAL PRIMERS EXTEND STRANDS 72oC • Each cycle of PCR doubles the number of progeny DNA duplexes (which can then act as template as well) • 1 cycle = 21 copies of starting template • 25 cycles = 225 copies of starting template 50 – 65oC

16. PCR Requirements Buffer – to maintain pH dNTPs – for incorporation into elongating DNA fragment MgCl2 – essential for primer binding 0.5 – 4 mM primers – forward and reverse pair gene specific 0.2 mM – 10 mM Enzyme – Taq polymerase or equivalent

17. 100 Conventional PCR Realtime qPCR 0 Cycle number 35 Quantitative PCR (qPCR) Regular PCR involves performing the reaction and electrophoresing the final product – not reflective of starting amount Machine measures amount of PCR product after each cycle by measuring the intensity of fluorescence Fluorescence from excitation of SYBR Green molecule by laser, SYBR Green only fluoresces when binds to dsDNA Real-time qPCR enables assessment of the reaction after each cycle Product Amount

18. 100 0 Cycle number 35 Quantitative PCR (qPCR) Create a standard curve with 10-fold serial dilutions of PCR product – assign arbitrary values Compare values from standards with values for unknown sample STD 1: 1,000,000 STD 2: 100,000 STD 3: 10,000 STD 4: 1,000 STD 5: 100 STD 6: 10 Product Amount Sample: 6,592

19. Hypothalamus Ob-R NPY -ve -ve -ve Adipocytes Regulation of Leptin Expression Leptin (Ob)

20. hOb gene structure Leptin mRNA in primary placental cultures Con FSK Coya et al. (2001) Biol. Reprod 65:814 Gong et al. (1996) JBC 271:3971 Regulation of Leptin Expression Human placenta also source of leptin BeWo cells (human choriocarcinoma) used as an in vitro model of placental function

21. Effect of Forskolin on BeWo Expression of Leptin 50 40 Relative Expression of leptin 30 20 10 0 Control Forskolin Regulation of Leptin Expression Treated BeWo cells with vehicle or forskolin for 72 hours Isolated total RNA from treated cells Reverse transcribed RNA to complementary cDNA – random hexamer primers Used Realtime RT-qPCR to determine leptin transcript levels Forksolin treatment increases leptin mRNA expression in BeWo placental cells 20 fold