Exam 3 Genetics

PCR purpose

amplifies a specific DNA fragment exponentially using heat-resistant DNA polymerase

PCR temperature steps

95 °C denature separates DNA strands then 55 °C anneal primers then 72 °C extend new strands with Taq polymerase

Taq polymerase origin

enzyme from Thermus aquaticus a hot-spring bacterium that is heat stable and survives repeated heating

PCR enzyme type

DNA polymerase not RNA polymerase

RT-PCR meaning

reverse transcriptase PCR used to convert mRNA into complementary DNA for expression analysis

Reverse transcriptase function

synthesizes complementary DNA from an RNA template

RT-PCR measures

mRNA levels which indicate gene expression rather than total DNA

qPCR purpose

quantitative PCR that uses fluorescence to measure DNA amount in real time

qPCR interpretation

earlier fluorescence curve means more starting DNA or cDNA and higher gene expression

Sanger sequencing principle

chain-termination DNA sequencing using fluorescent dideoxynucleotides that stop extension

ddNTP function

lacks a 3 prime hydroxyl group so once added the chain cannot continue growing

Sanger sequencing read length

produces about 500 to 1000 base pair reads of a single DNA fragment

Next-Generation Sequencing definition

massively parallel sequencing of millions of short DNA fragments at once without cloning

NGS read length

produces short reads about 150 base pairs but much faster and cheaper than Sanger

NGS key advantage

no bacterial cloning and can process entire genomes or transcriptomes simultaneously

NGS limitation

short reads make assembling repetitive regions difficult

Paired-end reads purpose

sequences both ends of a DNA fragment to determine distance and orientation for genome assembly

In situ hybridization purpose

uses labeled complementary RNA or DNA probes to show where in a tissue a gene is expressed

RT-PCR vs PCR difference

RT-PCR starts with RNA and measures expression while PCR only amplifies DNA

ddNTP nickname

dead-end nucleotide that terminates DNA synthesis in Sanger sequencing