DNA sequencing genes and genomes (Bio 172 exam 3)

sequencing definition

used to determine the nucleotide sequence of an unknown stretch of DNA, nucleotide by nucleotide

Reaction mix

• template DNA

• primer

• DNA polymerase

• deoxynucleotides

• labelled dideoxynucleotides

deoxynucleotide

has a hydroxyl (OH) group on the 3’ carbon, allowing this end to be elongated (unlabeled

dideoxynucleotide

lacks the 3’ hydroxyl group, can’t be elongated by DNA polymerase (all labeled)

dideoxy DNA sequencing

1) reaction mix with template and primer must be heated and cooled to allow primer to anneal

2) DNA polymerase is added and a single strand of DNA synthesis occurs

3) add C- will be terminated on some strands

• dCTP added: chain can be elongated

• ddCTP: chain terminated

4) will have DNA chains with range of sizes, each labeled with a flourescent dye

shotgun sequencing

1) cut genome into small fragments

2) sequence each fragment

3) assemble sequence

illumina sequencing

allows us to sequence millions of DNA fragments on a microscope slice

• also uses reversibly terminating nucleotides in sequencing reactions

nucleoid

loops of supercoiled DNA attached to a protein

chromatin packaging

• 10nm fiber to 30nm fiber

• composed of histone proteins: H2A, H2B, H3, H4

genome annotation

identifying various sequence features and their location in the genome

what is a reading frame

a way to rad nucleotide sequence on a genome

• 6 reading frames on a stretch of double stranded DNA

more proteins without more genes

• allternative splicing

• can be made from a single gene, depending upon how the RNA transcript is spliced together

more functions for each protein

1) combine proteins into different complexes

2) post translational modification of proteins to after function

C-value paradox:

no relationship between genome size and biological complexity

1) polyploidy: having more copies of the same gene

2) noncoding DNA: not all DNA codes for a protein

protein coding sequence definition

constitute about 2.5% of the human genome

repetitive sequence in eukaryotes

1) transposable elements

2) short tandem repeats

transposable elements definition

are parasitic segments of DNA able to move from one location in a genome to another

short tandem repeats

• microsatelites or simple sequence repeats, are repeating sequence of 1-5 bases

• minisatelites, or variable number terminal repeats, are repeating sequences of 6-500 bases