PCR + sequencing + cloning Flashcards
What are the necessary elements for Polymerase Chain Reaction?
- DNA template
- Taq pol (operates at high temp, comes from bacteria, no proofreading so can have errors, okk for short segments)
- DNA pair of Primers (one complementary to 5’ end, one for 3’ end)
- dNTPs (for amplification)
What are some uses for PCR?
- Sequencing
- DNA cloning (isolating particular gene)
- Detection of pathogens
- Gene editing
What does PCR rely on?
It relies on knowing the nucleotide sequence at the ends of the region to be amplified
What are the steps of PCR?
- Heat solution to 95˚C : Denature double helix (2 strands come appart, breaks H-bonds)
- Cool to 50˚C: primers anneal to DNA strands (excess amount of primers in initial solution)
2 types of primers: 3’ to 5’ and 5’ to 3’ - Raise T˚ to 70˚C: DNA synthesized with taq pol
(1st cycle synthesizes more than the sequence, but after segment ends so all good)
Repeat 1-3 for 40 cycles
- Final DNA extension: cool to bring single strands into double stranded helix again
How are produced oligonucleotides?
oligonucleotides = primers used in PCR
Designed by computer to be complementary and specific to end of sequence to be amplified + commercially synthesized
Computers make sure no other similar sequence in gene, have abt same ratio of AT/CG for both primers as want same T˚ of denaturation
What is the Dideoxycytidine’s Chain-Termination Method of DNA sequencing method (Classical Sanger Sequencing)?
Not so used anymore
4 tubes with:
DNA Polymerase
Oligonucleotide primer
DNA template
dNTPs (100 mM) of all 4 nucleotides
Each tube has 1 chain terminator:
ddATP (1mM)
ddGTP (1mM)
ddTTP (1mM)
ddCTP (1mM)
*has H on 3’ instead of OH so can’t make phosphodiester bonds
Use gel electrophoresis to figure out sequence
What are the limitations of Sanger sequencing?
Polymerase only runs for 300-500 nucleotides
Gells can only resolve this much so can only sequence short sequences
Rate of sequence of production limited by total number of reactions that can be performed at one time
Can only run about 40 cycles before lose too much precision
How does Next-Generation Sequencing (NGS) work?
*Single sequencing instrument carries out millions of sequencing reactions
One type:
1. Ligating linkers to mixture of DNA
2. Denature, then anneal to primers on solid support
PCR conducted to amplify DNA fragments in fixed spacial arrangement
- Double stranded DNA cut and only 1 strand sequenced with fluorescently labeled dNTPs (different colour/base)
For multiple DNA sequence at the time:
a. Add primer
b. Add fluorescently labeled dNTP which binds to single strand (just beside the primer)
c. Fluorescent imaging to determine which dNTP bound to which fragment
d. Chemically remove fluorophore (keep base pair)
e. Repeat until DNA strand is replicated, each base adds to next place beside
How is the whole genome assembled when doing Next-Generation Sequencing?
Computational models find overlaps in the fragments and align them to form a complete genomic sequence
What step is skipped in even newer sequencing methods than NGS?
PCR (sequencing is done on a single molecule)
motor protein moves through the DNA and send an electric signal to each base pair
Called nanopore
What are the advantages of Nanopore?
DNA sequencing technique with electric current (single molecule needed)
- Sequencing single molecules opens possibility of studying new biological questions
What are the general steps of the recombinant DNA technology?
- Vector + DNA fragment
- Recombinant DNA
- Replication of recombinant DNA within host cell
- Isolation, sequencing and manipulation of purified DNA fragment
What are plasmids?
- They are the most common vector used in recombinant DNA technologies
- Circular
- double-stranded DNA
- extrachromosomal (replicate independently from cell replication and division)
- found in bacteria and lower eukaryotes
- replication of plasmids occurs before cell division (not same amount of plasmids in every cells)
What are the roles of restriction enzymes (restriction endonucleases)?
Since the vector is ciruclar, have to cut it to insert DNA fragment, they cut phosphodiester bonds in vector (symmetrical fashion)
*Staggered cut to have sticky ends (not all rest. enzymes do, but better for further ligation)
Recognize a specific DNA sequence (Usually the sequence is a palyndrome to have same sticky ends)
Want 1 recognition site/vector
What is a polylinker?
It is a sequence with recognition sites for many different restriction enzymes
If sense of the DNA fragment matters, use 2 different restriction enzymes to have different sticky ends on each side
Polylinker = site where the exogenous DNA can be inserted
What are the 3 important parts of a Plasmid vector?
- Polylinker/recognition site
- Origine site
- drug resistant segment
What is the process of transformation of a Recombinant plasmid?
Process by which we isolate the transformed cells with recombinant and kill other cells
1. Mix E. coli with plasmids in presence of CaCl2, heat pulse (Recombinant plasmids entre the cell)
2. Culture on nutrient agar plates with ampicillin (cells without recombinant vector die)
3. Plasmid replication (before and independently from chromosome replication)
4. Cell multiplication (each cell has a different # recomb. vectors
What are DNA libraries?
Where permamnent collections of genes can be obtaied and maintained, collection of Recombinant vectors with different segments in them
- genomic libraries (chromosomal DNA, all DNA in the genome, mutliple different little segments?)
- cDNA libraries (represent mRNA present in a given sample)
How are cDNA libraries produced?
from RNA using reverse transcriptase (protein → RNA ← cDNA)