1. Techniques in Cloning

2. Polymerase Chain Reaction
Rapidly creates multiple copies of a segment of DNA
Uses repeated cycles of DNA synthesis in vitro
Used in DNA fingerprinting, kinship analysis, genetic testing for mutations, and infectious disease for diagnosis

3. PCR
Round 0 = 1 copy
Round 35 = billions of copies

4. PCR players DNA template – targeted piece of DNA
Primers – small segments of DNA that bind complementary upstream and downstream of the target on the template
Taq DNA polymerase – isolated from the Thermus aquaticus bacteria found in hotsprings of Yellowstone Park
DNA nucleotides in the form of deoxynucleoside triphosphates (dNTPs)
Reaction Buffer – maintains pH for enzymes

5. General PCR Process
Denaturation – split apart the two DNA strands by heating them to 95oC for 30s -1 min
Annealing – primers bind to target sequence by cooling reaction to 40-60oC for 30s - 1 min
Extension – Taq Polymerase extends the primers and copies each DNA template strand by heating to 72oC for 30s - 1 min

6. Primers
Required for both sides of the target sequence (forward & reverse primer)
Length of primer is generally nucleotides
G/C content and intra-complementarity are a concern when designing primers
Actually not a single primer for each but a mixture of primers (oligoprimers) if the sequence of the target is not known
If amino acid sequence of gene product is used then degenerate primers must be used
Initial forward primer is
GABTATGTTGTTGARTCTTCWGG
B=G/T/C R=G/A (purines) W =A/T

7. Nested PCR
Initial PCR primers are degenerate and based on a consensus sequence
The chances that the initial primers will bind to sequences other than the target are high
A second set of primers designed to be more specific to target is used
They are nested within the initial primers and are not degenerate thus much more specific to the target gene

8. Nested PCR

9. Our experiment Tube setup:
Add the following to a P3 tube (with PCR reaction pellet)
5 ul Target DNA template (P1)
10 ul Primer set (P2)
15 ul Enzyme -grade water (P4)
PCR Plan
Initial Denaturation 94oC for 5 minutes
Then 30 Cycles of:
Denaturation oC for 30 sec
Annealing oC for 30 sec
Extention oC for 30 sec
Final Extension oC for 5 minutes
Hold oC forever

10. Gel Electrophoresis
Definition: the process of separating molecules based on size and charge
Agarose: highly purified agar, heated and dissolved in buffer. Forms a matrix of pores for molecules to travel through.
Smaller molecules travel further
Molecules migrate towards the positive (red) end of the chamber

11. Gel Electrophoresis Process Make Agarose gel Prepare samples
Thinner gels (0.8%) yield better results for larger DNA
Prepare samples
Restriction enzymes used to cleave at specified sites
Apply samples to gels, apply current
If samples run from positive end they will run off the gel
Stain gels to see bands
Would not be able to see bands if we did not stain

12. Gel Electrophoresis DNA molecules have a negative charge
This allows them to migrate towards the positive end of the chamber
The samples and the electrophoresis chamber use specialized buffers. Using TAE/TBE buffer helps stabilize the sample and allows the reaction to occur quicker in the chamber.
If water were in the chamber instead of TAE/TBE buffer the reaction would take much longer or migration may not occur at all
Stains: ethidium bromide will cause the bands to glow orange under UV light. Fast stain will result in blue bands

13. Uses for Gel Electrophoresis
DNA fingerprinting or profiling
Paternity testing
Crime scene sample analysis
Identification of bacteria and other pathogens
Who is credited with discovering the DNA profiling process?
Alec Jefferies in 1985

14. Gel Electrophoresis

15. PCR purification
Small impurities can have a negative effect on the ligation of the PCR product to vector DNA
Impurities include unincorporated dNTPs, polymerases, primers and small primer-dimers.
A PCR spin column will remove the impurities in less than 4 min.

16. Restriction enzymes (endonucleases)
Definition: class of enzymes that cleave (cut) DNA at a specific and unique internal location along its length.
Makes 2 incisions, one through each of the sugar-phoshate backbones of the double helix
They can be naturally produced in bacteria and the bacteria use them as a defense mechanism against viral infection
The enzymes chop up the viral nucleic acids and destroy the virus
More than 3,000 known restriction enzymes
Common ones are: EcoRI, Psti, HindII

17. Restriction enzymes (endonucleases)
Discovered in late 1970s by Arber, Smith and Nathans
The chemical bonds that the enzymes cleave can be reformed by other enzymes known as ligases
Uses:
To insert new segment of DNA
To cut specific segments of DNA to study
To cut segment from one gene to insert it into another
Genetic engineering or recombinant DNA
Need suitable host, vector for carrying plasmid, way to get host to take up gene

18. Restriction Digests
Each enzyme cuts DNA at a specific sequence= restriction site
Many of the restriction sites are 4 or 6-base palindrome sequences
Enzyme cuts
Fragment 2
Fragment 1

19. Enzyme Examples EcoRI G-A-A-T-T-C C-T-T-A-A-G HindIII A-A-G-C-T-T
T-T-C-G-A-A
BamHI G-G-A-T-C-C
C-C-T-A-G-G
Bgl II A-G-A-T-C-T
T-C-T-A-G-A

20. Restriction enzymes (endonucleases)
Sticky ends: when unpaired length of bases (5’ AATT 3’) encounter an unpaired length of sequences (3’ TTAA 5’), they will bind or are “sticky” for each other.
Blunt ends: same length sequences or DNA section cut in half
Joining of two blunt ends is ligation

21. Restriction Digest Restriction Buffer provides optimal conditions:
NaCl provides correct ionic strength
Tris-HCl provides proper pH
Mg+2 is an enzyme co-factor
Body temperature (37oC) is optimal
Too hot kills enzyme
Too cool takes longer digestion time
Specific enzymes have specifc temps and times
Ody

22. Ligation
T4 DNA Ligase catalyzes formation of phosphodiesterase bond between 3’ hydroxy on one piece and the 5’ phosphate on another piece.
Requires ATP and Mg+2
Insert to vector DNA ratio should be 1:1
Proofing reading DNA polymerase removes dangling 3’A of PCR product

23. Products of Ligation Self-ligation of vector
Ligation of vector to primer-dimers
Ligation of multiple inserts
Self-ligation of inserts
Ligation of one insert into vector

24. Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA

25. Plasmids are good vectors:
small (2,000 – 10,000 bp)
circular, self-replicating
high copy number
multiple cloning sites (MCS)
selectable markers (Amp-resistance)
screening (reporter genes, positive select)
control mechanisms (lac operon)
can handle the size of the insert

26. Transformation
Once PCR product (insert) has been ligated into a plasmid, the plasmid be introduced into a living bacterial cell to replicate.
Two methods of transformation:
Electroporation
Heat Shock
Both methods make cells competent - able to take up plasmids

27. Transformation Steps Wash away growth media from cells
Place cells in ice cold calcium chloride which most likely hardens the cell membrane
Add plasmid to cells
Move cells to hot environment (usually 42oC) causes membrane pores to open so plasmid can enter
Add nutrient media to cells to allow them to recover from stress
Plate cells on selective growth plates (Amp and IPTG (increases expression of ampr gene)

28. Microbial Culturing
Pick a colony from the transformed cells to innoculate a liquid culture
Liquid culture (broth) must have selective antibiotic (Amp) in it.
Choose a single colony from the plate
Under favorable conditions, a single bacteria divides every 20 minutes and will multiply into billions in 24 hours

29. Plasmid Purification
To confirm that the engineered cells have been transformed with the correct DNA
Different methods
Lysozyme Method
Alkaline Cell Lysis Method
Column Methods (Aurum, EZNA)

30. Plasmid preps
Spectrophotometer determination of culture density. Take OD600 of culture (equal to about 8x108 cells/ml
Column can process up to 12 OD●ml of bacterial host cells
Cells disrupted with a lysis buffer
DNA binds to membrane of column, is washed and then eluted with aqueous buffer.

31. Restriction Digest Restriction Buffer provides optimal conditions:
NaCl provides correct ionic strength
Tris-HCl provides proper pH
Mg+2 is an enzyme co-factor
Body temperature (37oC) is optimal
Too hot kills enzyme
Too cool takes longer digestion time
Ody

32. Lambda DNA Lambda DNA comes from a bacteriophage
Genomic DNA of Lambda is well studied and used in research as a size markers for DNA pieces
Arrow mark HindIII restriction sites

33. DNA Sequencing
Determining the exact order of the nucleotide sequence in a DNA molecule.
Use to take days, now takes hours
Have sequences of entire genones for over 700 organisms

34. Sanger Method Prepare single-stranded DNA template to be sequenced
Divide DNA into four test tubes
Add primer to each tube to start DNA synthesis
Add DNA polymerase
Add labeled deoxynucleotides (dNTP) in excess. Labeled with radioactive or fluorescent tags
Add a single type of dideoxynucleotides (ddNTPs) to each tube. When incorporated in sythesized strand, synthesis terminates.
Allow DNA synthesis to proceed in each tube
Run newly synthesized DNA on a polyacrylamide gel

35. Reading the Sequence
In the tube with the ddTTP, every time it is time to add a T to the new strand, some Ts will be dTTP and some will be ddTTP.
When the ddTTP is added, then extension stops and you have a DNA fragment of a particular length.
The T tube will, therefore, have a series of DNA fragments that each terminate with a ddTTP.
Thus the T tube will show you everywhere there is a T on the gel
Same thing happens in all tubes
Read gel from top to bottom looking at all four lanes to get the sequence.

36. Automated Sequencing
Dye-terminator sequencing labels each of the ddNTPs with a different color fluorescent dye.
Now reaction can be run in one tube
Use capillary electrophoresis rather than the standard polyacrylamide slab gel.
When DNA fragment exits gel, the dyes are excited by a laser and emit a light that can be detected .
Produces a graph called a chromatogram or electopherogram

37. Automated Sequencing

38. Bioinformatics
Computerized databases to store, organize, and index the data and for specialized tools to view and analyze biological data
Uses include
Evolutionary biology
Protein modeling
Genome mapping
Databases are accessible to the public
Allow us to record, compare, or identify a DNA sequence

39. Types of RNA Messenger RNA (mRNA) Tranfer RNA (tRNA)
Ribosomal RNA (rRNA)
Signal Recognition Particle RNA (SRP RNA)
Small Interfering RNA (siRNA) – gene reg
Micro RNA (miRNA) – gene reg.

40. RNA Interference (RNAi)
Dicer enzyme cuts dsRNA up into smaller siRNA which then complex into the RNA-induced silencing complex (RISC) which then cuts up the mRNA
dsRNA can be engineered
so that genes can be
systematically shut down

41. Reverse Transcriptase PCR
Use reverse transcriptase
to make a DNA copy of
mRNA
Copy called cDNA
Allow scientists to study the
level of gene expression in
a cell

42. Northern Blots Run mRNA on a gel Transfer it to nitrocellulose
membrane
Add labeled cDNA probes to
the membrane and hybridize
the probes to the RNA
Allows you see what genes are expressed

43. DNA Microarray (Chip) Adhere genes to chip Collect mRNA from cells
Make labelled cDNA
from mRNA (red + green)
Add cDNA to chip
Measure signal