PCR Gel sequencing copy

Explain how to find the mutation caused by a gene

• 1. isolate and copy gene by adding specific DNA prime rthat binds (through complimentary base pairing) to either side of gene of interest through PCR

• 2. analyze amplified gene on a gel to determine length and expression level

• 3. sequence the insertion

• 4. compare sequence to known sequences to determine whats known about that region of the genome

What is PCR? Explain the process

• mimics DNA replication to create 10^6 copies of gene of interest

• 1. Heat DNA to separate strands (Mimics ORC)

• 2. mix DNA sample, DNA polymerase, synthetic DNA primers, dNTPs (nucleotides), and forward/reverse primers

• 3. cool strands slightly, allowing forward and reverse primers to anneal (bind to template)

• 4. heat to allow DNA synthesis

• repeat 25 cycles

Why do you amplify DNA?

• can analyze on a gel

• sequence

• clone

How does gel electrophoresis work? What infromation do you attain?

• negatively charged WT and mutant DNA placed on negative end of porous gel

• negatively charged DNA moves towards positive end of gel

• small DNA moves further through gel (its holey) and long DNA doesn’t move as far

• shows length of DNA; shows if theres an insertion/deletion causes a mutation

DNA Gel electrophoresis shows that the mutant doesn’t move as far as the wildtype. What does this mean about the mutant?

insertion in mutant relative to WT

How do homozygous and heterozygous alleles appear on DNA gel electrophoresis?

• homozygous: 1 band

• heterozygous: 2 bands

What do DNA gel and RNA gel detect and determine/compare?

• DNA gel - detects DNA fragment size; helps determine genotype

• RNA gel - detects mRNA abundance (expression level); tool fo rcomparing gene expressoin

What do a dark adn light band represent after RNA gel electrophoresis?

• dark: high expression

• light: less expression

RNA gel electrophoresis is performed. What do a dark, light, and no band in a mutant relative to the WT indicate?

• dark band: high expression - GOF

• light band : less expression - LOF (recessive)

• no band: low expression - LOF

What is different about a dideoxynucleotide triphosphate (ddNTP) used in Sanger sequencing from a normal dNTP? Why is ddNTP used in Sanger sequencing?

• does not have an OH on the 3’ C

• will not allow strand elongation once incorporated into a growing DNA stand

• not base-pair with the complementary strand

Describe Sanger sequencing

• Start with lots of PCR as template strand

• mix in lots of DNA template strands, synthetic primer, DNA polymerase, flourescently labeled ddNTPS, and more dNTPs

• thermocycle to denature, anneal, extend and repeat

• dNTPs/ddNTPS randomly incorporated by DNA poly

• results in strands of varying length with flourescently labeled ddNTPs corresponding to a specific nucleotide at the end

• line up fragments by size using gel electrophoresis, and follow the color to pattern to determine of nucleotides

What is a chromatogram?

• visual representation of the flourescence detected by the DNA sequencer

• each peak corresponds to 1 nucleotide

What is read length? What limits it?

• number of base pairs in a row that be sequenced in a single reaction

• DNA polymerase processivity (how long DNA poly can stay on before falling off)

What is a transposable element?

DNA region that moves around genome to change expression of a gene

Describe next-gen DNA sequencing, specifcially SMRT sequecning

• for sequencing an entire genome

• SMRT sequencing (single-molecule real time sequencing)

• long read length (high processsivity)

• accurate

• large outputs achieved by simultaneous analyis

• uses fluorescently labeled dNTPS (regular 3’ OH) for continuous syntehsis of strands and records color as DNA is lengthened

• results in a chromatograph with the order of nucleotides

• can do for RNA as well to see what genes are being expressed at that moment

Distinguish between replicating DNA and amplifying DNA, including:

• What performs the reaction and what’s being copied

• Which occurs in a cell and which occurs in vitro?

• What is the outcome of replication and amplification?

• DNA replication is performed by a complex of enzymes (DNA poly 3, primase, SSB, helicase, etc.), proceeds from origin of replication

• DNA amplification usually targets a specific gene/region of DNA and uses PCR, using heat stabilized taq-polymerases . uses heatcycling to denature, and help anneal and extend

• replication occurs within a cell, amplifying occurs in a lab (in vitro)

• replication results in two identical daughter DNA molecules, each containing one original strand and one new strand

• amplification results in millions of copies of a specific DNA fragment

What events occur at each different temperature during the polymerase chain reaction (PCR)?

• heating - denaturing/separating strands

• cooling - allows primers to anneal

• heating - allows DNA polymerase to extend

Sanger sequencing involves DNA polymerase, a primer, and nucleotides. What is special about the nucleotides used and how does this allow the DNA sequence to be determined (dideoxy, fluorescent)

• uses fluourescently labeled ddNTPs that lack a 3’ OH

• color corresponds to nucleotide

• can’t extend chain once ddNTPS added

How does SMRT technology sequence billions of base pairs in a single run of the machine? (output, read length, parallel)

• directly photographs/record DNA synthesis in real ime

• simultaneously records polymerase additions on multiple nucleotide fragments

• continuously synthesizing polymerase has long read length