genetics final 22/23/25

recombinant DNA

DNA from 2 different sources is combined

restriction enzymes

a specific piece of DNA that can be cut from genome

type II restriction enzymes

cuts at restriction site making staggered (sticky) or blunt cuts

gel electrophoresis

electricity in agarose gel causes DNA to move through gel (neg to pos)

what is DNA stained with after gel electro

ethidium bromide

southern blotting

confirm the presence, size, and abundance of a specific DNA sequence - done after electrophoresis

probe in southern blotting

single stranded piece of DNA complementary to sequence of interest - labeled with fluorescent tag or radioactivity

what can southern blotting be used to detect

homologous genes in different species

southern blotting process

purify DNA, cut DNA with restriction enzymes, run agarose gel, gel is moved to membrane , probe blot using ssDNA which binds to target DNA, autoradiography

Fluorescence in Situ Hybridization (FISH)

used to determine which chromosome a specific gene is on and to identify deletion or point mutation

FISH technique

metaphase chromosome spread is made, DNA is transferred to filter paper and denatured, incubate with fluorescently tagged probe

lack of probe in FISH

deletion mutation

Polymerase Chain Reaction

amplifies DNA of interest

PCR requirements

DNA template, DNAP (Taq), dNTPs, and primers

Why is DNAP Taq special

remains active at high temps

PCR process

melt DNA (90 C), cool to anneal primers (50 C), raise temp for dna synthesis (72 C)

genomic library

a collection of clones containing dna fragments from 1 genome

dna plasmids

used to carry DNA of interest into host cell - small, easy to purify, able to replicate, unique restriction sites, and antibiotic selective markers

how dna is inserted into plasmid

genomic DNA and plasmic DNA are cut with same restriction enzyme, combine cute (digested) DNA pieces, add ligase to seal recombinant plasmid

transformation

recombinant DNA is taken up by bacteria

blue white screening

sometimes not all plasmids take up target DNA; recombinant DNA results in disrupted lacZ gene and not produce b-gal. If lacZ produced b-gal, colonies turn BLUE and plasmid does not have target DNA. If lacZ does NOT produce b-gal, colonies are WHITE and plasmid is recombinant

purpose of amplicilin and tetracycline

plasmids contain tet and amp; recombinant plasmid only grows on amp media, empty plasmid only grows on tet and amp media

replica plating

one plate transformation reaction on amp media, a velvet surface is pressed onto the plate and transferred to replica plate with Tet media. Compare both plates and if any colonies disappear on replica plate it means bacteria has the target dna. Pick up correct colonies on amp plate

chromosome specific library

clones made from a specific chromosome

cDNA library

clones made from complementary DNA - synthesized from RNA using reverse transcriptase

properties of cDNA library

contains expressed sequences (no introns), useful for studying expression patterns in specific cell types

way to screen library - colony hybridization

• Grow bacterial colonies on a master plate.

• Transfer a copy of the colonies onto a nitrocellulose membrane.

• Lyse the bacteria on the membrane to expose their DNA.

• Add a labeled DNA probe

• The probe binds (hybridizes) to colonies that have your target DNA.

• Detect the signal - colonies that hybridized contain the gene of interest

way to screen library - expression of protein product

test bacterial colonies for enzymatic activity if protein function is known

way to screen library - positional cloning

uses probe for a gene NEAR the gene of interest and using chromosome walking to get the gene

chromsome walking

• Start with a known DNA sequence near the gene of interest.

• Clone a DNA fragment from that region.

• Sequence one end of that fragment to get a new known sequence.

• Use this new sequence to clone the next overlapping fragment.

• Repeat until the target gene is reached

physical mapping

distance based on direct analysis of DNA

what are ddNTPs used in Sanger method

it has no 3’OH which causes termination

sanger method

clone dna, synthesize labeled primer, denature DNA to get ssDNA, make mixture, divide mixtures into 4 tubes each with different ddNTPS, incubate, gel electrophoresis

contig map making

a collection of clones that contain OVERLAPPING pieces of chromosomal DNA

single nucleotide polymorphism (SNPs)

sites in genome where individual members of a species different by a single nucleotide due to mutations - inherit but do not show phenotype

haplotype

set of SNPs on a single chromosome

qualitative traits

discontinuous - only single gene and environment does NOT affect phenotype

quantitative

continuous - two or more genes and environment DOES affect phenotype

phenotype =?

genotype + environment

polygenic model of inheritance

more loci involved = more possible genotypes = more phenotypic classes

genetic-environmental interaction variation

environment can determine effect of genes and genes can determine effect of environment

additive variation

determines resemblance between parents and offspring

dominance variation

effect of 1 allele depends on identity of other allele

genic interaction variation

complementation, epistasis

heritability

variation in a phenotype is due to genetic differences among individuals

heritability conditions

specific for a particular population and time, heritability for a trait in 1 population will not be the same for that trait in another population

broad sense heritability

measures the proportion of total phenotypic variance due to all genetic factors

narrow sense heritability

Only the additive genetic variance which is the sum of individual allele effects that parents pass to offspring

artificial selection

how a phenotypic distribution will change if environment is modified

what does 0 value heritability means

none of variation is due to genotype variation and all variation is due to environmental variation

what does 1 value heritability means

all variation is due to genotypic variation

high h²

much of variation is due to genetic variation - trait should respond to selection

h_n² be used to determine what

selective breeding

what does R mean

response to selection changes after 1 generation of selective breeding

what does S mean

selectional differential: defines magnet of selection