BIO 1A03 T4M3: Applications of DNA replication

PCR + PCR set up

polymerase chain reaction

• sample DNA into buffered soln + DNA primers

  • primers: short, single-stranded DNA sequences that bind complementary to template DNA ==> serve as starting point for DNA copying (created with known GOI complementary sequences) 

• free dNTPs are added — so PCR doesn’t need all the enzymes for regular unwinding stabilizing, of DNA helix

  • building block of DNA (bbb nucleotides) 

• placed in thermocycler — 3 phases, repeated multiple times to amplify 

• tac polymerase: enzyme thats tolerates high temps, added to tube to catalyze polymerization of daughter strands 

stages of PCR

denaturation: high temp applied to denature (break H-bonds) to separate double strands

annealing: temp cools down, allow primers to anneal to complementary sequences on DNA template strands on either side of DNA of interest

• primers bind on opposite ends

extension: tac polymerase extends and polymerizes the daughter strand using 4 dNTPS (dATP, dCTP, dGTP, dTTP) from primer —> extends 5’-3’ DIR

what is the general formula of # of strands made from each round

2^n copies of template DNA sequence

gel electrophoresis

• separates DNA fragments (or RNA or proteins)

• moves thru porous gel and travels (-) to (+) [neg. inorganic phosphate towards the anode battery]

• able to separate a lot of fragments

factors at affect distance:

1) speed of molecules + size of molecules

• farther distance = faster = smaller molecules

• less distance = slow = bigger molecules

contains…

standardized ladder: contains DNA fragments of known sizes ==> allows for comparison to sample

dyes/stains: used to visualize nucleic acids/DNA fragments

sanger sequencing components 

• denatured single-stranded DNA

• primers

• free dNTPs

• DNA polymerase — catalyzes covalent bond btw 5’-P on one nucleotide to 3’-OH

ddNTPs

dideoxynucleotides

• missing OH group @ 3’ position — would not allow for further elongation bc an OH is required to attach to next nucleotide 

process of sanger sequencing

1) synthesis of each daughter strand begins @ 3’ of primer and continues until a ddNTP is randomly inserted 

• prevents further elongation 

2) each ddNTP is correlated to specific color of fluorscent (corresponding to the last added nucleotide) 

3) after synthesis > loaded into gel > fragments separated 

4) laser excites fluorescent dye

• detector records the amount of fluorescnets that emitted > matches it to one of the 4 ddNTP’s wavelengths 

• strands will differ by length bc it differs by 1 nucleotide 

5) spectrogram trace: produces peaks correlate to each ddNTP/fluorescent color thats tagged 

• allows to find the new daughter strands > to find template 

limitations = only good for short sequences 

shotgun sequencing + process

method for sequencing long DNA strands that involve breaking DNA into many small, random, overlapping fragments

1) amplify DNA

2) DNA fragments > cut into small pieces via enzymes 

3) analyase short sequences to see regions of overlapping nucleotides 

4) short sequences are put tog to generate long continous sequences of nucleotides 

5) computer/auto system will assembly overlapping DNA segments (contigs) to find whole genome 

• contigs: contiguous stretches of DNA sequences that are assembled from shorter, overlapping fragments 

6) Annotation (of genes) 

gene annotation

process of identifying location of genes and all of the coding regions in genome + function

• ** theres 6 possible reading frames in a double stranded DNA

• possible coding sequences: long seq of codons that lack codon (doesn’t stop prematurely)

  • looks for regulatory sites and promoters

  • looks for binding sites for TF’s (indicates upstream/downstream/in an intron of a gene)

  • looking for nearby complementary sequences == can infer the RNA forming hairpin

composition of functional genes in euk and prok

euk — exons/regulatory seq that code for functional genes = make up small % of genome (~50% consists of repeated non-coding seq)

prok — most of genome = functional genes