208 1st lecture

Dr. Michael Bulger, MRBX 1-11108 Dr. Yi-Tao Yu Dr. Peter Gibbs Series of lectures/short talks 3 write ups; schedule flexible depending on progress and other

commitments. 3 quizzes; READ AHEAD. One assignment. ASK Questions

EXPERIMENTAL OVERVIEW

PCR a DNA fragment using a plasmid template

Identify and purify the PCR product

Digest the product with suitable restriction enzymes

Ligate the product into a suitably digested cloning vector

Transform E. coli

Identify colonies containing the insert

Confirm the clone by miniprep of DNA and restriction digest

Perform large-scale DNA preparation

Sequence the chosen subclone

Analyze sequence by database searching/bioinformatics

PCR

aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta

Melting

94°C

PCR

aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat

ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta

Annealing

58°C

cgggcat

aactagg

PCR



Elongation

72°C

cgggcat

aactagg aatcgaatgtgcccgtacgattcgatgcga

gctaagctacgctttgatcctcgggatagcta

PCR



Melting

94°C

cgggcat



PCR



Annealing

58°C

cgggcat



cgggcat

cgggcat

aactagg

aactagg

PCR



Elongation

72°C

cgggcat



cgggcat

cgggcat

aactagg

aactagg

gcccgtacgattcgatgcga


aatcgaatgtgcccgtacgattcgatgcga

gctaagctacgctttgatcc



cgggcat


cgggcat

cgggcat





aactagg gcccgtacgattcgatgcga



cgggcat


cgggcat

cgggcat






cgggcat

aactagg

cgggcat

aactagg

cgggcat

aactagg

cgggcat

aactagg



cgggcat


cgggcat

cgggcat






cgggcat

aactagg

cgggcat

aactagg

cgggcat

aactagg

cgggcat

aactagg





aatcgaatgtgcccgtacgattcgatgcga




The PCR Amplification Plateau

Given that the individual components of a PCR reaction are not consumed and therefore do not become rate limiting, what is the cause of the amplification plateau ?

• Reagents unstable at high temps; Taq DNA polymerase has a finite half-life,

• Substrate excess; more DNA product present than remaining Taq can replicate in a given time,

• Incomplete template strand denaturation at high concentrations,

• Strand reannealing of specific product at high concentration prevents primer annealing,

• Competition for reactants by non-specific products-especially shorter products and primer dimers.

Hot Start PCR

During sample preparation at room temperature complexes of nonspecific primer-template or primer-primer may be generated. With HotStart a key component, such as primers, polymerase, Mg++, or dNTPs,necessary for amplification is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers.

In this way competing side reactions are minimized and a more specific amplification can occur.

• Add Taq manually to every tube when reaction reaches 94°C

• "Hot Wax" beads which contain MgCl2 emulsifed in paraffin beads.

• TaqBead, 1.25 ul/bead, facilitates hot start PCR by releasing Taq at 60°C

• Anti-Taq antibody; A heat labile antibody maintains Taq in an inactive state until ~60°C

Touchdown PCR

Touchdown PCR is a technique devised to minimize the impact of nonspecific annealing of primers to template during PCR. It takes advantage of the exponential amplification provided by PCR to favor the production of specific products. In this technique, the initial annealing temperature is set just below the calculated Tm for the primers. In a typical protocol, the annealing temperature is reduced by 1°C every 2 cycles (although there are many variations). Only the most specific primer-template interactions will take place at the highest annealing temperatures (likely to be at the sequence of interest), and so in later rounds, when the annealing temperature is lower, the specific products will “swamp out” the nonspecific products, even though all will be amplified.

Quantitative Real-Time PCR (qrt-PCR)

--qrt-PCR is a variation on PCR in which product amplification is measured quantitatively during cycling. --This is accomplished by detection of a fluorescent signal that is produced proportionally with PCR product. Example: SYBR-green

DNA intercalating dye, but only fluoresces when intercalated in double-stranded DNA. Thus, as cycling proceeds and the product is amplified, fluorescence can be measured during the annealing step.

Quantitative Real-Time PCR (qrt-PCR)

SYBR-green not specific - will detect all products, not just specific products. More specificity can be achieved using probe-based (i.e. “Taqman”) approaches.

ASSIGNMENT Find ANY paper that describes an application of PCR or uses PCR as a method for accomplishing the author’s experimental goals. In ~200 words describe the use of PCR in their particular experiment; the template, the primers (sequence, Tm [calculate it yourself if not stated] and other characteristics, the size of the products, how they were analyzed and the downstream application of their PCR products. Hand in next week i.e. 7 days time.

A. Determine thermal cycling parameters.!!1. Using the following guidelines, determine the thermal cycling parameters of the reaction and program the!thermal cycler. (Fill in the annealing temperature and extension times according to primer and product!considerations; see the pre-lab handout for help). Run the program for 30 cycles.!!Denature ___ min at ___°C !!Anneal ___ min at _____ °C:!!If primer GC content is <=50%, anneal at 55°C; if >50%, anneal at 60°C. Also, calculate the Tms of the!two primers using the '2+4' rule to assess the validity of the above guideline.!!Primer 1; ___°C; Primer 2; ___°C.!!Extend _____ min at ___°C:!!If product length is <=500 nucleotides, extend 1 min; if >500 nucleotides, extend 3 min.!!Your product is anticipated to be ~500bp.!!!Lengthen the time for the last extension step of the last cycle to 7 min, to ensure that all the PCR products!

!are full-length.!

0.5 94 0.5 50

52 56 1.5 72

208 1st lecture

Education

Transcript of 208 1st lecture