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

Gel Electrophoresis (1/2)

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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!!

Some genes this is already done

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

454 FLX

Pyrosequencing

IlluminaSolexa Sequencing

http://www.youtube.com/watch?feature=player_detailpage&v=yVf2295JqUg

Nanosequencing

SMRT Sequencing