BIO 210 Chapter 4

Steps of DNA replication

1. DNA unwinds at the origin of replication 2. helicase opens up the DNA-forming replication forks; these are extended bidirectionally 3. single strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA 4. topioisomerase binds at the region ahead of the replication fork to prevent supercoiling 5. primase synthesizes RNA primers complementary to the DNA strand 6. DNA polymerase starts adding nucleotides to the 3' -OH end of the primer 7. elongation of both the lagging and the leading strand continues 8. RNA primers are removed by exonuclease activity 9. Gaps are filled by DNA pol by adding dNTPs 10. the gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds

In vitro DNA synthesis not only help determine the sequence of DNA but also used to _______ amount of DNA present in a sample

amplify

Overlap PCR

can be used to link two different gene segments. In this scheme, the overlapping primer has one end with sequences complementary to target sequence 1, and the other half similar to target sequence 2. The PCR reaction will create a product with these two regions linked together allows strands from two different fragments to hybridize to one another. This forms an overlap. When this overlap is extended by DNA polymerase yields a recombinant molecule.

telomerase

extends telomeres, preventing their degradation, in some cell types.

Reverse transcriptase PCR

uses mRNA as a template to convert it back to complementary DNA (cDNA) using the enzyme reverse transcriptase. The cDNA is then amplified by PCR. This technique is particularly useful when working with eukaryotic DNA, since most eukaryotic genes contain introns that are removed from the primary transcript before becoming mRNA. Working backward prevents interference of intron sequences. Uses the enzyme reverse transcriptase to make a cDNA copy of the mRNA from an organism and then uses PCR to amplify the cDNA

Some of the differences exist between prokaryotes, eukaryotes, and other genetic elements (viruses, plasmids) in terms of replication. what is it???

In bacteria, there is usually only one ori to initiate replication of the typical single, circular chromosome

Degenerate primers PCR

It's a combination of oligonucleotide sequences in which few bases are altered in such a way that the primer covers the all possible nucleotide combinations for the targeted protein through the DNA sequence developed by working backward from a protein sequence to a possible nucleotide sequence based on the genetic code. these primers are mixtures of oligonucleotides that have two or three different bases in the third position of the codon

directed mutagenesis

PCR can be used to make small changes in nucleotide sequences mutation of a single or multiple nucleotides in the DNA at a specific location

Creating artifical restriction sites by PCR

PCR has revolutionized molecular biology and biotechnology. Genes can be amplified using primers containing engineered restriction sites so that the PCR product can be digested with the restriction enzyme and then cloned into a vector.

What direction does in vivo DNA synthesis take place?

3' to 5' direction

Chain Termination Method of Sequencing

DNA Polymerase randomly stops at each nucleotide. The mix is run on a gel; the shortest fragments run fastest.

In prokaryotes, three main types of polymerases are known:

DNA pol I, DNA pol II, DNA pol III

Forensic analysis of tiny samples at a crime scene and identification of infectious diseases prior to the emergence of symptoms both use PCR

TRUE

Why is RNA primer NOT used in in vitro synthesis of DNA?

RNA is very unstable and degrades easily. instead, a short single-stranded oligonucleotide of DNA is used as a primer.

DNA pol II

Repair function

DNA Ligase

Seals the gaps between the Okazaki fragments to create one continuous DNA Links the 3'-OH to the 5'-PO4 forming the phosphodiester bridge.

rolling circle replication

a DNA replication mechanism in which one strand is nicked and unrolled for use as a template to synthesize a complementary strand Replication of circular DNA that is initiated by a break in one of the nucleotide strands, producing a double-stranded circular DNA molecule and a single-stranded linear DNA molecule, the latter of which may circularize and serve as a template for the synthesis of a complementary strand. Unidirectional replication of a circular DNA molecule like a plasmid that involves nicking one DNA strand and displacing it while synthesizing a new strand

Synthesizing the lagging strand

because the lagging strand is synthesized in small pieces, either DNA polymerase I or RNase H excise the RNA bases and replace them with DNA. DNA ligase closes the nicks in the sugar/phosphate backbone of the new DNA strand

Next generation sequencing

use a solid-surface platform that can hold millions of DNA fragments in separate locations. After DNA from the organism of interest is isolated, the first step in next-generation sequencing is to amplify the DNA using PCR and then add linkers or adapters to the DNA fragments by cloning them into TA vectors

TA cloning vector

used to insert the products of PCR directly into the vector without the use of restriction enzymes. Taq polymerase has Terminal transferase can add A to the 3' end These DNA fragments can be cloned in vector that has 5' overhang "T" - no specific restriction sites are needed.

Problems for replication at the ends due to linear nature of Eukaryotic chromosomes

ends shorten after each round of replication

Sliding Clamp

helps hold the DNA polymerase in place when nucleotides are being added

inverse PCR

is a variant of the polymerase chain reaction that is used to amplify DNA with only one known sequence. uses a restriction enzyme to cut DNA in a region of unknown sequence to aid in the sequencing of the unknown region next to a known region.

S phase

only point within eukaryotic cell cycle where they replicate their genomes

Illumina sequencing

the fragments are attached to a flow cell. once the fragments are attached to the platform, PCR is used again to create multiple copies of the single pieces of DNA fluorescent dyes for each base are used, and wavelengths are monitored by computers that translate the information into a DNA sequence

454 sequencing

the fragments are attached to the solid surface via beads linked to the adapter flashes of light are produced whenever a base is added. this information is sent to a computer to translate into a base identity

function of taq polymerase in PCR

to amplify or synthesise DNA or gene of interest for various downstream application. It's a type of thermostable DNA polymerase, can work at higher temperature as well."

PCR can be used to delete, insert, and even fuse different gene segments

true

Catenated

two daughter copies are linked together like links in a chain

topoisomerase IV

untangles the two chromosomes so they can partition into the daughter cells

DNA gyrase

-Untwists strands of DNA. -removes the supercoiling

replication fork

Y-shaped region of DNA

Two most common types of next generation sequencing

-454 sequencing -Illumina sequencing

3 steps in each cycle of PCR

-DENATURATION (of the DNA into single-stranded pieces (94°C) -ANNEALING the primers to the DNA (50°C-60°C) -ELONGATION new DNA using the polymerase and the 3′-OH provided by the primers (72°C).

DNA pol I

-Exonuclease activity removes RNA primer and replaces with newly synthesized DNA -removes the RNA primers from the lagging strand

DNA pol III

-Main enzyme that adds nucleotides in the 5'-3' direction. -main enzyme involved in replicating DNA -requires a 3'-OH to be able to start replicating

mismatch repair system

-identifies the mistake -determines which strand is the original (presumably the correct one) -removes the incorrect nucleotide -inserts the correct one using the original strand as a template

Helicase

-opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases -unwinds double helix

In vitro DNA synthesis requires...

-single stranded template DNA -DNA polymerase -oligonucleotide primer -nucleotide precursors (supplied as dATP, dTTP, dCTP, dGTP, collectively known as dNTPs)

Primase

-synthesizes RNA primers needed to start replication -initializer -DNA polymerase can't figure out where to get started without primer -primase makes the primer so DNA polymerase can figure out where to start -primer is made of RNA

Components of a PCR reaction

-template DNA -dNTPs -set of primers -taq polymerase -mix buffer -pcr tube -thermocycler

cycle sequencing mixes

-template DNA -primer -taq polymerase -deoxynucleotides (dATP, dTTP, dGTP, dCTP) - dideoxyribonucleotides (ddATP, ddTTP, ddGTP, ddCTP)

Single stranded binding proteins (SSB)

Binds to single stranded DNA to avoid DNA rewinding back

Topoisomerase

Helps relieve the stress on DNA when unwinding by causing breaks and then resealing the DNA keeps DNA from supercoiling

difference between illumina and 454

The two methods differ by how exactly the DNA is amplified, but the end result is the same: Clusters of identical DNA fragments are produced.