2.2 analyzing and manipulating dnaPresentation Transcript
Analyzing &Manipulating DNA
WHAT IS GENETIC ENGINEERING?
Special Cases: Selective Breeding• Artificial selection: breeding only those plants or animals with desirable traits• People have been using selective breeding for 1000’s of years with farm crops and domesticated animals.
Hybridization The crossing of dissimilar individuals to bringtogether the best of both organisms, but genomic incompatibility usually leads to sterility.
Donkey Horse Or does it? Mule female horse + male donkey male horse + female donkeyfemale mule + male donkey = sterile offspring Hinny female mule + male horse = male stud While there is no known instance of a male mule siringoffspring, female mules have on very rare occasion given birth to viable offspring. Most of the offspring of these female mules have been sterile, except on stallion that was completely reverted.
Genetic Bottlenecks & Founder EffectsCan be likened to Artificial Selection, hybridization, and inbreeding
InbreedingThe continued breeding of individuals with close relation to one another. Inbreeding rapidly purifies the genome by exposing homozygous recessives.
Basic steps in genetic engineering1. Isolate the gene2. Insert it in a host using a vector3. Produce as many copies of the host as possible4. Separate and purify the product of the gene5. Manipulate it any way you want
Vectors are our canvasesBasic vector musts:1 Origin of Replication(phyllum specific)2 Ability to selectfor/against3 Multiple cloning site
Cloned Enzymes!!!Polymerase – make new DNA or RNA againstan existing DNA or RNA templateLigase – joins two DNA or RNANuclease – cleaves into twoDNA Repair Proteins – self-explanatoryMethyltransferase – adds methyl groupsPhosphatase - removes 5´ phosphate groupsfrom DNA and RNAKinase– adds 5´ phosphateRecombinase – Swap strands in and out
Polymerases copy DNA and RNATaq DNA Polymerase is thermostablepolymerase that elongates chains 5´→ 3´Klenow Fragment goes 5´→ 3´T7 RNA Polymerase synthesizes RNA inthe 5´→ 3´ direction from DNA templatecontaining a T7 phage promoter.
Ligases glue pieces together
Nucleases cut DNAExonuclease I – binds single-stranded;removes primers in a polymerase reactionmixtureExonuclease III – binds double-stranded;chews back 3´-5´; can control the numberremoved.
Nuclease EndonucleasesType I endonucleases were the discovered first. bind at one place but cut a random distance (c.a.1000 bp) away. Cleavage follows a process of DNA translocation, which shows that these enzymes are also molecular motors. asymmetrical recognition site is and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a non-specific spacer of about 6–8 nucleotides. These enzymes are multifunctional and are capable of both restriction and modification activities, depending upon the methylation status of the target DNA.
TYPE II endonucleasesAside from PCR and primers, ‘restriction enzymes’ are the major workhorses in our toolkit.
Most RE Recognition Sequences are Palindromes EcoRI BamHI Bg1II NofIG^AATT-C G^GATC-C A^GATC-C GC^GGCC-GCC-TTAA^G C-CTAG^G T-CTAG^G CG-CCGG^CG
PCR – Polymerase Chain Reaction Making copies to work with.
PCR Cloning Primer Design Specific primers Degenerated primers Nested primers Amplification High-fidelity DNA polymerase Hot start Touch down PCR Clone into appropriate vector Compatible restriction sites Poly T (pGEM T easy) No ligation (Topo cloning)
Primer Design Considerations Primers must be specific for desired sequence to be amplified primers should be long enough to ensure specificity (usually 18-30 bases) primers normally screened against databases Primers must form stable duplex at annealing temperature No complementarity between forward and reverse primers or primers and product
Initial primer selection criteria Length (18-25 bases) Base composition (45-55% GC) Melting temperature (55-80 C) 3’ terminal sequence strong bonding base (G or C) at end no runs (3 or more) of G or C at end
Primer complementarity criteria Primer vs. self & forward vs. reverse maximum number of consecutive bonds maximum number of consecutive G-C bonds Forward primer vs. Reverse primer maximum number of consecutive bonds between the 3’ ends Primer vs. product maximum number of consecutive bonds between the 3’ ends
PCR amplificationPCR amplification cycles consist of 3 main steps:a. Denaturing step leading to strand separation which occurs at high temperature (usually over 80°C and typically 94-95 °C). The denaturing step usually lasts for 15 to 30 sec but can be extended for long fragments of template DNA and Hot Start Polymerases (2-3 min).b. Annealing step corresponds to primer hybridization with the template. The annealing temperature is dependent on the primer sequences and base composition and usually is kept lower than 72°C. Annealing time of 30-45 seconds commonly used. Increasing annealing time does not drastically influence the outcome of PCR reactions- DNApol has reduced activity @ 45- 65°C, thus longer annealing times may increase the likelihood of nonspecific amplification. Tm of primer in °C = (no. of Gs and Cs x 4) + (no. of As and Ts x 2) Ideally the Tm of each primer (forward and reverse) should be similar (±2 °C) The optimal annealing temperature to reduce NSB is usually (Tm – 5 °C)c. Extension step is normally 72°C for NPRs and 68 °C for PRs; duration normally 1 min for every 1 Kb to be amplified (depends on the DNApol used). *Usually a final extension at 72°C for 5 to 10 min is included at the end of the cycle to allow the completion of the extension of all the products.
What if all you know is the protein sequence?
Examplea protein motif: W D T A G Q E Trp Asp Thr Ala Gly Gln Glu5 TGG GAY ACN GCN GGN CARGAR 3where the Y = C or T, R = G or A, N = G, A, T or C.(This gives a mix of 256 differentoligonucleotides.)
What if youdon’t even know the protein sequence?
Degenerate Primers for Discovery P P P P P P P
Degenerate Primers for Discovery P P P P P P PV /A V/S K PL V/G P A SGUN GUN AAA CCN UUA GUN CCN GCN AGUGCN UCN AAG UUG GGN AGC CUNGYN KYN AAR CCN YTN GKN CCN GCN AGY 24 224 2 4 2 4 24 4 4 2 = 262,144 SEQUENCES!!
Some genes this is already done
Touch Down / Step down A one-step procedure for optimizing PCRsIt involves the use of anannealing temperature that ishigher than the target optimumin early PCR cycles. Theannealing temperature isdecreased by 1°C every cycle orevery second cycle until aspecified or touchdownannealing temperature isreached.This only works on paralogs ifthere are sequence differences 45- 55- 55in the “primed” sequence. 55 45
NESTED PCR A powerful method to amplify specific sequences of DNA from a large COMPLEX mixture of DNA. Overcomes non-specific amplification [even paralogs*] by using two sets of primers (almost like using a longer primer).* Primer Design is crucial!
DNA Sequencing1. By separation (Sanger)2. By synthesis 1. Polymerase vs. Ligase 2. fluorophore vs. pyrophosphate 3. Polony vs. multiplexing3. Nanopore