Post on 15-Jun-2015
description
transcript
Analyzing & Manipulating
DNA
WHAT IS GENETIC ENGINEERING?
• Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.
• Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.
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.
Selective breeding
HybridizationThe crossing of dissimilar individuals to bring
together the best of both organisms, but genomic incompatibility usually leads to sterility.
Or does it?
While there is no known instance of a male mule siring offspring, 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.
male horse + female donkey
Donkey Horse
Mule
Hinny
female horse + male donkey
female mule + male donkey = sterile offspring
female mule + male horse = male stud
Genetic Bottlenecks &
Founder Effects
Can be likened to Artificial Selection, hybridization, and inbreeding
Inbreeding
The continued breeding of individuals with close relation to one another.
Inbreeding rapidly purifies the genome by exposing homozygous recessives.
Genetic Engineering
Basic steps in genetic engineering
1. 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 canvases
Basic vector musts:1 Origin of Replication (phyllum specific)2 Ability to select for/against3 Multiple cloning site
Cloned Enzymes!!!Polymerase – make new DNA or RNA against an existing DNA or RNA templateLigase – joins two DNA or RNANuclease – cleaves into twoDNA Repair Proteins – self-explanatoryMethyltransferase – adds methyl groupsPhosphatase - removes 5´ phosphate groups from DNA and RNAKinase– adds 5´ phosphateRecombinase – Swap strands in and out
Polymerases copy DNA and RNA
Taq DNA Polymerase is thermostable polymerase that elongates chains 5´→ 3´ Klenow Fragment goes 5´→ 3´ T7 RNA Polymerase synthesizes RNA in the 5´→ 3´ direction from DNA template containing a T7 phage promoter.
Ligases glue pieces together
Exonuclease I – binds single-stranded; removes primers in a polymerase reaction mixture
Exonuclease III – binds double-stranded; chews back 3´-5´; can control the number removed.
Nucleases cut DNA
Type 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.
NucleaseEndonucleases
TYPE II endonucleasesAside from PCR and primers, ‘restriction enzymes’
are the major workhorses in our toolkit.
Most RE RecognitionSequences are Palindromes
G^AATT-CC-TTAA^G
G^GATC-CC-CTAG^G
A^GATC-CT-CTAG^G
GC^GGCC-GCCG-CCGG^CG
EcoRI BamHI Bg1II NofI
Mor
e R
E E
nzym
esEnzyme Sequence Product
EcoRI G^AATTC 5’ sticky ends
BamHI G^GATCC 5’ sticky ends
Bg1II A^GATCT 5’ sticky ends
PvuI CGATC^G 3’ sticky ends
PvuII CAG^CTG Blunt end
MboI G^ATC 5’ sticky ends
HindIII A^AGCTT 5’ sticky ends
HinfI G^ANTC 5’ sticky ends
Sau3A G^ATC 5’ sticky ends
AluI AG^CT Blunt end
TaqI T^CGA 5’ sticky ends
HaeIII G^GCC 5’ sticky ends
NofI GC^GGCCGC 5’ sticky ends
DNA Fingerprinting
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Gel Electrophoresis
Gel
Ele
ctro
phor
esis
(2/
2)
Methyltransferases
Phosphatases
Kinases
Recombinases
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 (55-80C) 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 amplification PCR 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?
Degenerate Primers
Examplea protein motif:
W D T A G Q E Trp Asp Thr Ala Gly Gln Glu 5' TGG GAY ACN GCN GGN CAR GAR 3'
where the Y = C or T, R = G or A, N = G, A, T or C.
(This gives a mix of 256 different oligonucleotides.)
What if you don’t even know the protein
sequence?
Degenerate Primers for Discovery
PPPPPPP
Degenerate Primers
Degenerate Primers for Discovery
PPPPPPP
V /A V/S K P L 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!!
Touch Down / Step down A one-step procedure for optimizing PCRs
45-55
55-45
55
It involves the use of an annealing temperature that is higher than the target optimum in early PCR cycles. The annealing temperature is decreased by 1°C every cycle or every second cycle until a specified or 'touchdown' annealing temperature is reached. This only works on paralogs if there are sequence differences in the “primed” sequence.
NESTED PCRA 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. pyrophosphate3. Polony vs. multiplexing
3. Nanopore
PCR-based Dye terminator nucleoside Massive capillary electrophoresis
Sanger Sequencing