Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
30 Cards in this Set
- Front
- Back
|
What is obtained from DNA sequencing? |
The sequence of bases in a polypeptide |
|
|
What is the most commonly used DNA sequencing method? |
The Sanger method |
|
|
What is the basis of the Sanger Method? |
Uses DNA polyemerase to copy single stranded DNA Makes use of the fact that DNA polymerase stops when it reaches a dideoxynucleotide so the chain is terminated |
|
|
What are dideoxynucleotides? |
Do not have an OH on the 3' carbon of the nucleotide |
|
|
Method for dideoxynucleotide sequencing |
Small amount of each bases relivent ddNTP is added to the DNA This allows the DNA to be terminated when it reaches that specific base The products from the reaction are run on seperate lanes on a polyacrimide gel which allows seperation according to length The base sequence can then be visualised using X-ray film |
|
|
What is automated DNA sequencing? |
Uses a single reaction for each DNA sequence in which all four ddNTPs are added Each ddNTP is labelled with a differently coloured flourescent marker The DNA fragments are seperated on a capilary gel As the fragments move down the gel it passes a laser which excites the flourescent tag on each fragment as it passes which creates a graoh showing the bases |
|
|
Why can the flourescent markers identify specific bases? |
Because they each have a different wavelength so can be identified |
|
|
Method for large scale sequencing |
Use restriction enzymes to produce map of DNA region Fragment DNA into small pieces Clone into vector Sequence DNA clones using primer in vector Use mapping data and sequence overlap between clones to align sequence from fragments to get complete sequence |
|
|
How many bases does original radioactive sequencing sequence per reaction and how long does it take? |
200-400bp, takes days |
|
|
How many bases does flouresence sequencing sequence per reaction and how long does it take? |
800-1000bp, takes hours |
|
|
What is next generation sequencing? |
uses sequential addition of nucleotides of microchips to sequence DNA |
|
|
How many bases does onext generation sequencing sequence per reaction and how long does it take? |
50-200 but takes seconds |
|
|
What is bioinformatics? |
Uses computational techniques to organise, share and analyze sequence information |
|
|
Functions of structural and functional genomics |
Identifying features in the genome Charactorise gene structure Predict gene regulatory regions predict gene functions Identify gene clusters and families |
|
|
What is comparitive genomics? |
Comparing genome sequences between species |
|
|
Functions of comparitive genomics |
Identify sequences conserved over species Establish phylogenetic relationships between species Identify conserved chromosomal regions Stufy evolution of genes Examine relationships between genomes and organisms environment |
|
|
What is PCR? |
a powerful technique used to allow selective amplication of specific regions of DNA |
|
|
What type of DNA polymerase is used in PCR? |
Taq polymerase |
|
|
Why is Taq polymerase used in PCR? |
It can withstand high temperatures without denaturing |
|
|
Is PCR a sensitive process? |
Yes, it can amplify DNA from very small amounts |
|
|
What are the three steps to PCR? |
Denaturation Primer anealing Primer extension |
|
|
How much DNA is copied per sample? |
The number of copies is doubled per sample |
|
|
What happens in the denaturation step of PCR? |
Heated to around 95C DNA is denatured into single strands |
|
|
What happens in the primer annealing step of PCR? |
Temperature lowered to 45-68C Primers hybridize to their complementary sequences on the target DNA |
|
|
What happens in the primer extension step of PCR? |
Temp raised to 72C Allows Taq polymerase to synthesize DNA |
|
|
How are the products of PCR analyzed? |
Seperated by gel electrophoresis |
|
|
Limitations of PCR |
Must know some information about the nucleotide sequence of target DNA to synthesise the primers Minor contamination of sample can cause problems Cannot amplify long segments of DNA |
|
|
Uses of PCR |
Amplify genes from genetic DNA or cDNA for use in cloning or analysis Can amplify specific pieces of DNA from variey of samples Identify victims in natural disasters |
|
|
What is reverse transcription PCR? |
Used to study gene expression by examining mRNA produced by cells or tissues |
|
|
What is quantitative real time PCR? |
allows researchers to quantify amplication reactions as they occur in real time to identify amount of DNA in a sample |
