Lecture 3: Tools of molecular biology and their application to genotyping

Why is DNA’s negative charge important for molecular biology

DNA can be seperated using its charge, such as with gel electrophoresis

what happens when DNA is denatured

the two DNA strands separate

what happens when DNA is renatured

complementary DNA strands come back together

What is a primer

a short sequence that allows DNA polymerase to begin copying a specific DNA region

What do restriction enzymes do

cut DNA at specific nucleotide sequences

what is gel electrophoresis used for

separating and visualizing DNA fragments based on size

how does DNA move through an agarose gel

DNA migrates through the gel when an electrical charge is applies

how does DNA fragment size affect migration through a gel

smaller fragments migrate faster; larger fragments migrate more slowly

what is a DNA size standard (ladder)

A mixture of DNA fragments of known sizes used to estimate fragment sizes

Why is stain used in gel electrophoresis

to make DNA fragments visible as bands

What is PCR used for

making many copies of a specific piece of DNA

How do PCR primers target a specific DNA region

primers are designed to bind to each end of the target sequence

what can amplified using PCR

variable sites or genes of interest

what is DNA sequencing used for

determining the sequence of bases in DNA

how does Sanger sequencing work

ddNTPs stop DNA synthesis when incorporated, allowing the DNA sequence to be determined

what is a ddNTP

a modified nucleotide that stops DNA synthesis when incorporated

How does illumina/next-generation sequencing differ from Sanger sequencing

it sequences millions of DNA fragments simultaneously, producing much higher throughput

what is a sequencing library

a collection of randomly fragmented DNA prepared for next-generation sequencing

why is bioinformatics important for next-generation sequencing

heavy computation is needd to interpret the millions of sequences produced

what is plymorphism

a genetic diffrence or variant among individuals of a species

what is genotyping

determining an individuals allele(s) at a genetic location

alleles are

different versions of a gene or homologous DNA region

what does diploid mean

having two copies of each gene, one on each parental chromosome

homozygous

having two identical alleles at a genetic location

heterozygoys

having two different alleles at a genetic location

genetic marker

a historical term for a DNA variant used to identify genetic differences

does a genetic marker have to be associated with a gene

no. it may be in a gene, near and gene, or nowhere near a gene

what are two broad types of molecular polymorphisms

single base-pair polymorphisms and repetitive sequence polymorphisms

what makes a useful genetic marker for conservation research

it should be easy/cheap to genotype, polymorphic, and co-dominant

what does polymorphic mean for a genetic marker

more than one allele is segregating in the population or species

what does co-dominant mean for a genetic marker

it can distinguish both homozygotes and the heterozygotes

what are the major types of molecular markers

isozymes, AFLPs, microsatellites, organelle markers, and SNPs

what are the key molecular biology tools used for genotypying

gel electrophoresis, PCR, restriction digestion, and DNA sequencing