biol 2210 exam 3

genomic clone

clone of an entire genome

restriction enzymes

enzymes that cut as specific palindromic sequences (read the same on each strand). Come from bacteria, defend against viruses

sequence specific primer

primer specific to a nucleic acid sequence. makes sure that the wanted sequence is copied. useful in PCR

amplification

use of PCR to get more DNA to work with (increasing quantity)

plasmid

extra bacterial DNA. able to put any DNA into it to make recombinant DNA

clone

a group of genetically identical individuals derived from (and identical to) a common ancestor

sticky end

result from RE with overhangs at the cleavage site (ex. EcoRI)

blunt end

no overhang from a RE site

recombinant DNA

DNA containing DNA from 2 different organisms. no homology other than overhang

how to estimate restriction enzyme frequency

P=0.25 for any given bp. (1/4)^#bp of enzyme cut sequence.

homology

having the same structure. Other than the overhang sites, recombinant DNA has no homology

cloning vector

a replicating genetic element used to clone (purify and amplify) a DNA segment in a living cell
must have: multiple cloning sites (to be able to insert foreign DNA), Ori, and a selectable marker

selectable marker

a gene that allows you to tell if a vector has incorporated donor DNA. can select for it, it marks the vector. ex ampicillin resistance gene

DNA library

clone of all fragments of donor DNA. created by cleaving a DNA sequence with a RE, as well as a clone vector. Ligase anneals sticky overhangs to insert donor DNA into vector. Uses a marker gene to make sure that DNA hybridized.

polylinker

many restriction enzyme sites within a marker gene. If the marker gene does not function, you know there was insertion

genomic DNA library

entire of collection of clones for a species, each with a different segment of genomic DNA.

cDNA

complementary DNA. Cloned DNA from mRNA in a cell. Only expressed DNA

cDNA library

library of all the expressed DNA in an organism

primer

a short single stranded RNA or DNA that acts as a start site for in vitro synthesis when bound to a single stranded template

oglionucleotide

short segment of synthetic DNA

RNA dependent DNA polymerase

DNA polymerase that is able to use RNA as a template for DNA synthesis. Essentially a reverse transcriptase

dsDNA

double stranded DNA

linkers

primers that are blunt on the end, allows for subcloning of each small piece of cDNA.

transformation

method of delivering recombinant DNA into bacterial cells

transduction

using a phage to deliver a plasmid to bacterial cells

infection

infecting bacteria with actual virus/phage DNA/RNA. ends up with viral DNA produced

how to find specific DNA clones in a library

using nucleic acid probes from a cDNA clone/population, purified mRNA, homologous gene from related organism, protein product.

can also use an antibody against a specific protein

cDNA probe

- label known cDNA with radioactive substance
- plate recombinant DNA on agar medium
- replicate colonies to nylon membrane
- lyse cells in situ, bind DNA to membrane
- ssDNA from colonies are on plate
- add radioactive cDNA under annealing conditions
- remove non-annealed cDNA
- expose to x-ray film; dark spots will be where matching DNA is
- pick DNA colony from plate to isolate DNA of interest

restriction map

map of all the restriction enzyme cut sites on a given plasmid/piece of DNA

southern blot

using gel electrophoresis to probe DNA. DNA is cut, then transferred a membrane. after being labeled with a probe, it is exposed to x-ray film. some bands are darker due to higher levels of cDNA binding.
- tests for size and number of genomic DNA that hybridize to the probe

northern blot

tests for size of mRNA transcript, and whether mRNA is expressed in a cell or tissue.

western blot

tests for size of protein and whether the protein is expressed in a given cell/tissue type

probe

labelled nucleic acid segment used to identify specific nucleic acid molecules bearing the complementary sequence.

PCR

polymerase chain reaction
cycling of 3 reactions: denaturation of DNA template and primers at 94C. Annealing of primers to DNA template at 54C. Extension, reading of DNA template by polymerase at 72C.

- add oglionucleotides, heat to separate strands, cool to anneal primers
- heat to allow DNA synthesis
- repeat

taq polymerase

heat resistant polymerase that allows it to be heated multiple times. reduces need to manually add more primer and polymerase

Klenow DNA pol 1

more stable version of DNA pol 1, for use in PCR

DNA sequencing

determining the sequence of DNA through several methods. Uses DNA polymerase

- from e coli, make an extract, remove nucleic acids, remove DNA using DNase, purify with proteins
- with proteins, add a buffer, Mg2+, dNTPs, DNA template, DNA primer, enzyme
- makes a radioactive polymer

dNTP

radioactively tagged nucleotide

ddNTP

dideoxynucleotide. DNA pol requires a 3'-OH group to extend the nucleotide chain, but dideoxys don't have this, so chain stops.

Sanger sequencing

using ddNTPS to sequence a DNA sequence. some ddNTPs (only one type at a time to keep nucleotides separate) are mixed with dNTPs. Where ddNTPs incorporate instead of dNTPs, the chain stops. When there are a bunch of various length chains (due to synthesis stopping only when ddNTPs are incorporated). When run on a gel, the farther the piece goes the smaller it is, and the closer to the beginning of the chain it is.

automated DNA sequencing

uses sanger method, but ddNTPs are each tagged a different fluorescing color. When run through a computer, it detects the wavelength and thus the sequence. Can sequence 121,600 bp/day

structural genomics

used if a genome doesnt have many repeats (i.e. E. coli).
aka shotgun sequencing
- a genome and its copies are cut into many fragments
- overlapped sequence reads
- overlap contains for complete sequence

contig

series of overlapping DNA sequences used to make a physical sequence map

GAPS

repetitive sequence that cannot be spanned by sequencing and are not in unique locations within the genome (repeat is present)

paired end reads

used to join two sequence contigs
- when the repeat is between contigs A and B, each end of the repeat will be cloned from a long-insert vector. Uses primers to the vector sequence to sequence. It is not necessary to sequence through the middle to link A and B contigs

human genome length

3.08x10^9 bp (3.08 Gb).
most of the genome is euchromatin

euchromatin

chromosomal region that is lightly packed chromatin and contains transcribed genes

heterochromatin

tightly packed chromatin that is mostly genetically inert (like telomeres, centromeres, some genes). Low gene density and highly repetitive

ORF (open reading frame)

large sections of DNA that are transcribed. usually the coding portion of genes.

how to find binding sites in genes

can program sequence into computer, where it will predict binding sites based off other known sites, other organisms. It will also predict splice sites

BLAST similarity

compares DNA sequences for similarity across species

codon bias

some organisms tend to encode a particular degenerate codon over another (ex. using CCU over CCC for proline). This makes it difficult to compare genes across species

chromosomal landscape

map of where coding vs noncoding regions are in a genome

annotated sequence map

shows where individual genes are on a chromosome

homologous genes

genes derived from a common ancestral gene. If two genes are homologous, it makes it easier to predict function.

orthologous genes

genes in different organism that share a common ancestral gene (ex actin genes)

paralogous genes

genes in the same organism that have evolved by gene duplication (ex olfactory receptor genes)

cytogenic map

stains chromosomes. Used in FISH, measured in um

physical map

maps restriction sites (kb)

recombination map

maps meiotic recombination (uses cM -- recombination frequency determines #)

sequence map

map of Sanger sequencing; actual DNA sequence (kb)

molecular marker

ex. micro satellites.
Things on the gene that identify it from other genes
general mms: STS, EST
allelic mms: RFLP, VNTR, SNP

general molecular marker

tell where you are in the genome. universal (doesnt vary person to person)

allelic molecular marker

differentiate between chromosomes and vary individual to individual. Can help track phenotypes

STS

sequence tagged site. 200-500bp that has a single occurrence in the human genome and whose location and base sequence are known.

detectable by PCR, help with orienting mapping and sequence data from different labs

EST

expressed sequence tags, STSs derived from cDNA. defines a gene being expressed. when cDNA libraries are used to sequence these, if sequences are longer than 300 bps, 2 identical primers are needed to sequence both ends

RFLP

restriction fragment length polymorphism. variation between individuals in DNA fragment sizes cut by specific REs; polymorphic sequences that result in RFLPs are used as markers, usually caused by alterations at a restriction endonuclease cutting site.

If an SNP change introduces a new RE site, it can be tracked amongst individuals via restriction digests and southern blots

useful for when phenotype is

VNTR

variable nucleotide tandem repeat. exhibit allelic variation, detected via a probe. used in DNA fingerprinting. a southern blot will detect all VNTRS in a sequence. vary heavily between individuals

type of DNA polymorphism. Number of repeats differs across individuals

SNP

single nucleotide polymorphism. if it lands within the restriction site, you can tell if it's been cut and can follow it. Alters one base pair.

silent SNPs can tag a gene, they can be associated with a mutant phenotype, intergenic SNPs can be used on markers (though there is no known effect), RFLPs are SNPs are located at restriction enzyme sites

Tandem Repeat polymorphisms

helpful due to the repetitive nature of the genome. gives us info about different chromosomes.

microsatellite markers

very small TRPs, ~1-6 bp repeated about 50-100x. co-segregates in a pedigree with a dominant disease allele. so polymorphic that you can tell the differences between the mother and father chromosome. Is linked to the gene, but not part of the actual gene.

length is correlated with the allele.

mini satellite markers

small TRPs, ~7-14 bp repeated 5-50x

single base pair allelic distinctions

used to distinguish between individuals

SNP clusters

clusters of SNPs, useful in distinguishing between individuals

PCR primer

primer used in PCR, with specific sequence in order to PCR a certain chain of nucleotides

DNA polymorphism

one of two or more alternate forms (alleles) of a chromosomal locus that differ in nucleotide sequence or have variable numbers of repeated nucleotide units

combining PCR and SNP analysis for sickle-cell anemia

beta globin is the earliest protein to be sequenced. They got DNA sequence and looked for restriction sites. Mutation of Glu>Val eliminates a RE cut site. Use MstII to cut PCRd DNA, find genotype of parents and children to determine if children have SCA.

transposable element

DNA sequences of various lengths and sequence that can move within the genome. If a TE lands within a gene, it can inactivate it. They increase genome size (via copy and paste transposons). Thought to be junk, but some transposable elements contain expressed genes that may help organisms. They can help drive gene expression in new patterns

transposition

the process by which a transposable element moved

Barbara McClintock

- studied chromosome breakage in maize
- one cross where chr9 frequently broke, it led to sterile, lethal, and unexplained spotted kernels.
- the C gene causes pigmentation in corn. when the Ds element is inserted into the gene, it stops coloration. When Ds is removed from only some cells (in the presence of Ac), it causes spotted kernels. When Ac is in the gene, it also causes spotted cells.

Ds

Dissociation factor. non-autonomous element in maize. When in the C gene, no transcription happens. When paired with the Ac element, it is able to jump out of the genome, causing repaired function to C gene and allowing color to be made

Ac

Activator. autonomous element in maize. Codes for its own transposes, when by itself can jump in and out of genes, making spotting. Required for mobilization of Ds.

Sometimes, alleles of Ac turn into Ds allele and no longer has movement function

autonomous transposon

a transposon that encodes its own transposase. Is able to move without the presence of anything else

non-autonomous transposon

a transposon that does not encode its own transposase. requires another, autonomous transposon to move around

insertion sequences

short mobile elements with imperfect inverted terminal repeats, encode onto protein necessary for mobilization

transposon

types of transposable elements

Class II transposons (DNA elements)

elements that don't use mRNA as an intermediate to incorporate into a genome. Conservative replication, have varying levels of complexity. staggered at cut site and have target site duplication

Class I transposons (retrotransposons)

elements that use mRNA as an intermediate to insert into genome. Starts as RNA then inserts itself. Replicative. Derived from retroviruses. copies transposon already in genome into RNA, encodes reverse transcriptase, then copies itself back into another site in DNA

replicative transposon

replicate themselves and insert the new copy into a new site. Net gain of one transposon (how genomes can get huge)

conservative/cut-and-paste transposon

mobilizing a transposon from one site to another (physically cut out using transposase enzyme that they encode). constant number of these in the genome

retrotransposon

transposition occurs through a process that involves insertion of DNA copies of the element from an RNA intermediate

terminal inverted repeats

elements usually have repeats at the end. important to insertion. encodes transposase to put into genome. when insertion happens, they cut jaggedly for a target site duplication

target site duplication

duplication of the nucleotides on either side of the target site, due to jagged cuts that are repaired by DNA repair mechanisms

composite transposon

encode another gene alongside transposase. Derived from 2 insertion sequences that mobilized together and took the DNA between them. can form inverted repeats. this is how bacteria gain genes.

simple transposon

also encodes transposase, can also take genetic material between two inverted repeats. MUST be flanked by 2 repeats

resistance factors

F-like factors (plasmids) that contain transposons with drug resistance genes. can spread resistance through bacteria via conjugation

retrotransposons have features of retroviruses

- insert into genome, can't get rid of it unless you destroy cells
- packages itself up to insert into another cell
- tend to have repeats, reverse transcriptase, and integrate
- thought to have originated from retroviruses

LINE

long interspersed element. autonomous transposition, very large fraction of human genome (21%). retrotransposon

SINE

short interspersed element. non autonomous transposition, 13% human genome. retrotransposon

drosophila p-element

useful for transgeneis. first transposable element molecularly characterized and harnessed for cloning and transformation. Inverted repeats, encodes transposase. introduced into lab flies by wild flies in early 1900s. can use these to carry genes into a genome